Filovirus Consensus Antigens, Nucleic Acid Constructs and Vaccines Made Therefrom, and Methods of Using Same

ABSTRACT

Nucleic acid molecules and compositions comprising one or more nucleic acid sequences that encode a consensus Ebolavirus glycoproteinimmunogens are disclosed. The coding sequences optionally include operable linked coding sequence that encode a signal peptide. Immunomodulatory methods and methods of inducing an immune response against Ebolavirus are disclosed. Method of preventing Ebolavirus and methods of treating individuals infected with Ebolavirus are disclosed. Consensus Ebolavirus proteins are disclosed.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.62/332,372, filed May 5, 2016, U.S. Provisional Application No.62/402,519, filed Sep. 30, 2016, and U.S. Provisional Application No62/483,979, filed Apr. 11, 2017, each of which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to vaccines for inducing immune responsesand preventing filovirus infection and/or treating individuals infectedwith filovirus, particularly infection by Ebolavirus. The presentinvention relates to consensus Ebolavirus proteins and nucleic acidmolecules which encode the same.

BACKGROUND OF THE INVENTION

The Filoviridae are non-segmented, single stranded RNA viruses whichcontain two divergent genera, Marburgvirus (MARV) and Ebolavirus (EBOV).Members from each can cause severe and highly lethal hemorrhagic feverdisease to which there is no cure or licensed vaccine (Bradfute S. B.,et al. (2011) Filovirus vaccines. Hum Vaccin 7: 701-711; Falzarano D.,et al. (2011) Progress in filovirus vaccine development: evaluating thepotential for clinical use. Expert Rev Vaccines 10: 63-77; Fields B. N.,et al. (2007) Fields' virology. Philadelphia: Lippincott Williams &Wilkins. 2 v. (xix, 3091, 1-3086 p.); Richardson J. S., et al. (2009)Enhanced protection against Ebola virus mediated by an improvedadenovirus-based vaccine. PLoS One 4: e5308; and Towner J. S., et al.(2006) Marburgvirus genomics and association with a large hemorrhagicfever outbreak in Angola. J Virol 80: 6497-6516).

Due to lethality rates of up to 90% they have been described as “one ofthe most virulent viral diseases known to man” by the World HealthOrganization. The US Centers for Disease Control and Prevention hasclassified them as ‘Category A Bioterrorism Agents’ due in part to theirpotential threat to national security if weaponized (Burki T. K. (2011)USA focuses on Ebola vaccine but research gaps remain. Lancet 378: 389).These ‘high priority’ agents could in theory be easily transmitted,result in high mortality rates, cause major public health impact andpanic, and require special action for public health preparedness (CDC(2011) Bioterrorism Agents/Diseases. Atlanta: Centers for DiseaseControl and Prevention).

The haemorrhagic fever diseases are acute infectious with no carrierstate, although they are easily transmissible among humans and nonhumanprimates by direct contact with contaminated bodily fluids, blood, andtissue (Feldmann H., et al. (2003) Ebola virus: from discovery tovaccine. Nat Rev Immunol 3: 677-685). During outbreak situations, reuseof medical equipment, health care facilities with limited resources, anduntimely application of prevention measures escalate transmission of thedisease, allowing amplification of infections in medical settings.

Since the natural reservoirs of these zoonotic pathogens are likely tobe African bats and pigs (Kobinger G. P., et al. (2011) Replication,pathogenicity, shedding, and transmission of Zaire ebolavirus in pigs. JInfect Dis 204: 200-208), the latter possibly being more of anamplifying host, the manner in which the virus first appears at thestart of an outbreak is thought to occur through human contact with aninfected animal. Unpredictable endemic surfacing in the Philippines,potentially Europe, and primarily Africa of this disease furtherconstitutes a major public health concern (Outbreak news. (2009) EbolaReston in pigs and humans, Philippines. Wkly Epidemiol Rec 84: 49-50).

Experiments have been performed to determine the capacity of the vaccinefor inducing protective efficacy and broad CTL including experiments inrodent preclinical studies. (Fenimore P W, et al. (2012). Designing andtesting broadly-protective filoviral vaccines optimized for cytotoxicT-lymphocyte epitope coverage. PLoS ONE 7: e44769; Hensley L E, et al.(2010). Demonstration of cross-protective vaccine immunity against anemerging pathogenic Ebolavirus Species. PLoS Pathog 6: e1000904; Zahn R,et al (2012). Ad35 and ad26 vaccine vectors induce potent andcross-reactive antibody and T-cell responses to multiple filovirusspecies. PLoS ONE 7: e44115; Geisbert T W, Feldmann H (2011).Recombinant vesicular stomatitis virus-based vaccines against Ebola andMarburg virus infections. J Infect Dis 204 Suppl 3: S1075-1081; andGrant-Klein R J, Van Deusen N M, Badger C V, Hannaman D, Dupuy L C,Schmaljohn C S (2012). A multiagent filovirus DNA vaccine delivered byintramuscular electroporation completely protects mice from ebola andMarburg virus challenge. Hum Vaccin Immunother 8; Grant-Klein R J,Altamura L A, Schmaljohn C S (2011). Progress in recombinant DNA-derivedvaccines for Lassa virus and filoviruses. Virus Res 162: 148-161).

Vaccine-induced adaptive immune responses have been described innumerous preclinical animal models (Blaney J E, et al. (2011).Inactivated or live-attenuated bivalent vaccines that confer protectionagainst rabies and Ebola viruses. J Virol 85: 10605-10616; Dowling W, etal. (2007). Influences of glycosylation on antigenicity, immunogenicity,and protective efficacy of ebola virus GP DNA vaccines. J Virol 81:1821-1837; Jones S M, et al. (2005). Live attenuated recombinant vaccineprotects nonhuman primates against Ebola and Marburg viruses. Nat Med11: 786-790; Kalina W V, Warfield K L, Olinger G G, Bavari S (2009).Discovery of common Marburgvirus protective epitopes in a BALB/c mousemodel. Virol J 6: 132; Kobinger G P, et al. (2006). Chimpanzeeadenovirus vaccine protects against Zaire Ebola virus. Virology 346:394-401; Olinger G G, et al. (2005). Protective cytotoxic T-cellresponses induced by Venezuelan equine encephalitis virus repliconsexpressing Ebola virus proteins. J Virol 79: 14189-14196; Rao M, Bray M,Alving C R, Jahrling P, Matyas G R (2002). Induction of immune responsesin mice and monkeys to Ebola virus after immunization withliposome-encapsulated irradiated Ebola virus: protection in micerequires CD4(+) T cells. J Virol 76: 9176-9185; Rao M, Matyas G R,Grieder F, Anderson K, Jahrling P B, Alving C R (1999). Cytotoxic Tlymphocytes to Ebola Zaire virus are induced in mice by immunizationwith liposomes containing lipid A. Vaccine 17: 2991-2998; Richardson JS, et al. (2009). Enhanced protection against Ebola virus mediated by animproved adenovirus-based vaccine. PLoS One 4: e5308; Vanderzanden L, etal (1998). DNA vaccines expressing either the GP or NP genes of Ebolavirus protect mice from lethal challenge. Virology 246: 134-144;Warfield K L, et al. (2005). Induction of humoral and CD8+ T cellresponses are required for protection against lethal Ebola virusinfection. J Immunol175: 1184-1191; Jones S M, et al. (2007). Assessmentof a vesicular stomatitis virus-based vaccine by use of the mouse modelof Ebola virus hemorrhagic fever. J Infect Dis 196 Supp12: S404-412Grant-Klein R J, Van Deusen N M, Badger C V, Hannaman D, Dupuy L C,Schmaljohn C S (2012). A multiagent filovirus DNA vaccine delivered byintramuscular electroporation completely protects mice from ebola andMarburg virus challenge. Hum Vaccin Immunother 8.; Geisbert T W, et al.(2010). Vector choice determines immunogenicity and potency of geneticvaccines against Angola Marburg virus in nonhuman primates. J Virol 84:10386-10394.) Viral vaccines have shown promise and include mainly therecombinant adenoviruses and vesicular stomatitis viruses.Non-infectious strategies such as recombinant DNA and Ag-coupledvirus-like particle (VLP) vaccines have also demonstrated levels ofpreclinical efficacy and are generally considered to be safer thanvirus-based platforms. Virus-specific Abs, when applied passively, canbe protective when applied either before or immediately after infection(Gupta M, Mahanty S, Bray M, Ahmed R, Rollin P E (2001). Passivetransfer of antibodies protects immunocompetent and immunodeficient miceagainst lethal Ebola virus infection without complete inhibition ofviral replication. J Virol 75: 4649-4654; Marzi A, et al. (2012).Protective efficacy of neutralizing monoclonal antibodies in a nonhumanprimate model of Ebola hemorrhagic fever. PLoS ONE 7: e36192; Parren PW, Geisbert T W, Maruyama T, Jahrling P B, Burton D R (2002). Pre- andpostexposure prophylaxis of Ebola virus infection in an animal model bypassive transfer of a neutralizing human antibody. J Virol 76:6408-6412; Qiu X, et al. (2012). Ebola GP-Specific Monoclonal AntibodiesProtect Mice and Guinea Pigs from Lethal Ebola Virus Infection. PLoSNeglTrop Dis 6: e1575; Wilson J A, et al. (2000). Epitopes involved inantibody-mediated protection from Ebola virus. Science 287: 1664-1666;Sullivan N J, et al. (2011). CD8(+) cellular immunity mediates rAd5vaccine protection against Ebola virus infection of nonhuman primates.Nat Med 17: 1128-1131; Bradfute S B, Warfield K L, Bavari S (2008).Functional CD8+ T cell responses in lethal Ebola virus infection. JImmunol 180: 4058-4066; Warfield K L, Olinger G G (2011). Protectiverole of cytotoxic T lymphocytes in filovirus hemorrhagic fever. J BiomedBiotechnol 2011: 984241). T cells have also been shown to provideprotection based on studies performed in knockout mice, depletionstudies in NHPs, and murine adoptive transfer studies where efficacy wasgreatly associated with the lytic function of adoptively-transferredCD8+ T cells. However, little detailed analysis of this response asdriven by a protective vaccine has been reported.

Countermeasure development will ultimately require an improvedunderstanding of protective immune correlates and how they are modulatedduring infection. This proves difficult when infected individuals whosuccumb to filoviral disease fail to mount an early immune response.These fast-moving hemorrhagic fever diseases result in immunedysregulation, as demonstrated by the lack of a virus-specific Abresponse and a great reduction in gross T cell numbers, leading touncontrolled viral replication and multi-organ infection and failure.Conversely, survivors of Ebola virus (EBOV) disease exhibit an early andtransient IgM response, which is quickly followed by increasing levelsof virus-specific IgG and CTL. These observations suggest that humoraland cell-mediated immune responses play a role in conferring protectionagainst disease. These data are also supported by numerous preclinicalefficacy studies demonstrating the contribution of vaccine-inducedadaptive immunity to protection against lethal challenge. However,mounting evidence has demonstrated a critical role for T cells inproviding protection where efficacy was greatly associated with thefunctional phenotype of CD8+ T cells. While these recent studieshighlight the importance of T cells in providing protection, theirprecise contributions remain uncharacterized and controversial.Furthermore, little detailed analysis of this response driven by aprotective vaccine has been reported.

SUMMARY OF THE INVENTION

Isolated nucleic acid molecules comprising on or more nucleic acidsequences encoding a a first consensus Zaire ebolavirus envelopeglycoprotein immunogen (ZEBOVCON), a nucleic acid encoding a secondconsensus Zaire ebolavirus envelope glycoprotein immunogen (ZEBOVCON2),or a nucleic acid encoding a ZEBOV Guinea 2014 Outbreak envelopeglycoprotein immunogen (ZEBOVGUI) are provided.

In one embodiment, the the ZEBOVCON comprises an amino acid sequencethat is at least 95% homologous to SEQ ID NO:1, a fragment of an aminoacid sequence that is at least 95% homologous to SEQ ID NO:1, an aminoacid sequence that is at least 99% homologous to SEQ ID NO:1, a fragmentof an amino acid sequence that is at least 99% homologous to SEQ IDNO:1, an amino acid sequence of SEQ ID NO:1, or a fragment of SEQ IDNO:1.

In one embodiment, the ZEBOVCON2 comprises an amino acid sequence thatis at least 95% homologous to SEQ ID NO:68, a fragment of an amino acidsequence that is at least 95% homologous to SEQ ID NO:68, an amino acidsequence that is at least 99% homologous to SEQ ID NO: 68, a fragment ofan amino acid sequence that is at least 99% homologous to SEQ ID NO: 68,an amino acid sequence of SEQ ID NO: 68, or a fragment of SEQ ID NO: 68.

In one embodiment, the ZEBOVGUI comprises an amino acid sequence that isat least 95% homologous to SEQ ID NO:67, a fragment of an amino acidsequence that is at least 95% homologous to SEQ ID NO:67, an amino acidsequence that is at least 99% homologous to SEQ ID NO: 67, a fragment ofan amino acid sequence that is at least 99% homologous to SEQ ID NO: 67,an amino acid sequence of SEQ ID NO: 67, or a fragment of SEQ ID NO: 67.

In some embodiments, the fragments comprise at least 600 amino acids, atleast 630 amino acids, or at least 660 amino acids.

In one embodiment, ZEBOVCON is linked to an IgE signal peptide. In oneembodiment, ZEBOVCON2 is linked to an IgE signal peptide. In oneembodiment, ZEBOVGUI is linked to an IgE signal peptide.

In one embodiment, the nucleic acid encoding ZEBOVCON comprises anucleic acid sequence at least 95% homologous SEQ ID NO:69, or afragment thereof. In one embodiment, the nucleic acid encoding ZEBOVGUIcomprises a nucleic acid sequence at least 95% homologous SEQ ID NO:72,or a fragment thereof. In one embodiment, the nucleic acid encodingZEBOVCON2 comprises a nucleic acid sequence at least 95% homologous SEQID NO:70, or a fragment thereof.

In one embodiment, the nucleic acid encoding ZEBOVCON comprises anucleic acid transcribed from a DNA sequence at least 95% homologous SEQID NO:69, or a fragment thereof. In one embodiment, the nucleic acidencoding ZEBOVGUI comprises a nucleic acid transcribed from a DNAsequence at least 95% homologous SEQ ID NO:72, or a fragment thereof. Inone embodiment, the nucleic acid encoding ZEBOVCON2 comprises a nucleicacid transcribed from a DNA sequence at least 95% homologous SEQ IDNO:70, or a fragment thereof.

A composition comprising one or more nucleic acid sequence encoding oneor more of ZEBOVCON, ZEBOVCON2 and ZEBOVGUI is also provided. In oneembodiment, the composition comprises two or more nucleic acid sequenceencoding two or more of ZEBOVCON, ZEBOVCON2 and ZEBOVGUI. In oneembodiment, the composition comprises two nucleic acid molecules. In oneembodiment, the composition comprises three or more nucleic acidsequence encoding ZEBOVCON, ZEBOVCON2 and ZEBOVGUI. In one embodiment,the composition comprises three nucleic acid molecules.

The invention also provides novel sequence for producing immunogens inmammalian cells or viral vectors.

A composition comprising a nucleic acid sequence that encodes aconsensus Zaire ebolavirus envelope glycoprotein immunogen, a nucleicacid sequence that encodes a consensus Sudan ebolavirus envelopeglycoprotein immunogen, and a nucleic acid sequence that encodes aMarburg marburgvirus Angola 2005 envelope glycoprotein immunogen isprovided. The amino acid sequence of the consensus Zaire ebolavirusenvelope glycoprotein immunogen may be SEQ ID NO:1 (ZEBOV CON), afragment of SEQ ID NO:1, an amino acid sequence that is homologous toSEQ ID NO:1, or a fragment of an amino acid sequence that is homologousto SEQ ID NO:1. Amino acid sequences that are homologous to SEQ ID NO:1are typically 95% or more, 96% or more, 97% or more, 99% or more, or 99%or more, homologous to SEQ ID NO:1. Fragments of SEQ ID NO:1 orfragments of amino acid sequences that are homologous to SEQ ID NO:1 aretypically 600 or more, 630 or more, or 660 or more amino acids. Theamino acid sequence of the consensus Sudan ebolavirus envelopeglycoprotein immunogen may be SEQ ID NO:2 (SUDV CON), a fragment of SEQID NO:2, an amino acid sequence that is homologous to SEQ ID NO:2, or afragment of an amino acid sequence that is homologous to SEQ ID NO:2.Amino acid sequences that are homologous to SEQ ID NO:1 are typically95% or more, 96% or more, 97% or more, 99% or more, or 99% or more,homologous to SEQ ID NO:2. Fragments of SEQ ID NO:2 or fragments ofamino acid sequences that are homologous to SEQ ID NO:2 are typically600 or more, 630 or more, or 660 or more amino acids. The amino acidsequence of the Marburg marburgvirus Angola 2005 envelope glycoproteinimmunogen may be SEQ ID NO:3 (MARV ANG), a fragment of SEQ ID NO:3, anamino acid sequence that is homologous to SEQ ID NO:3, or a fragment ofan amino acid sequence that is homologous to SEQ ID NO:3. Amino acidsequences that are homologous to SEQ ID NO:3 are typically 95% or more,96% or more, 97% or more, 99% or more, or 99% or more, homologous to SEQID NO:3. Fragments of SEQ ID NO:3 or fragments of amino acid sequencesthat are homologous to SEQ ID NO:3 are typically 600 or more, 637 ormore, or 670 or more amino acids. The amino acid sequence may optionallycomprise a leader sequences such as the IgE leader.

A composition comprising a nucleic acid sequence that encodes aconsensus Zaire ebolavirus envelope glycoprotein immunogen, a nucleicacid sequence that encodes a consensus Sudan ebolavirus envelopeglycoprotein immunogen, a nucleic acid sequence that encodes a Marburgmarburgvirus first consensus envelope glycoprotein immunogen, a nucleicacid sequence that encodes a Marburg marburgvirus second consensusenvelope glycoprotein immunogen, and a nucleic acid sequence thatencodes a Marburg marburgvirus third consensus envelope glycoproteinimmunogen is also provided. The amino acid sequence of the consensusZaire ebolavirus envelope glycoprotein immunogen may be SEQ ID NO:1(ZEBOV CON), a fragment of SEQ ID NO:1, an amino acid sequence that ishomologous to SEQ ID NO:1, or a fragment of an amino acid sequence thatis homologous to SEQ ID NO:1. Amino acid sequences that are homologousto SEQ ID NO:1 are typically 95% or more, 96% or more, 97% or more, 99%or more, or 99% or more, homologous to SEQ ID NO:1. Fragments of SEQ IDNO:1 or fragments of amino acid sequences that are homologous to SEQ IDNO:1 are typically 600 or more, 630 or more, or 660 or more amino acids.The amino acid sequence of the consensus Sudan ebolavirus envelopeglycoprotein immunogen may be SEQ ID NO:2 (SUDV CON), a fragment of SEQID NO:2, an amino acid sequence that is homologous to SEQ ID NO:2, or afragment of an amino acid sequence that is homologous to SEQ ID NO:2.Amino acid sequences that are homologous to SEQ ID NO:1 are typically95% or more, 96% or more, 97% or more, 99% or more, or 99% or more,homologous to SEQ ID NO:2. Fragments of SEQ ID NO:2 or fragments ofamino acid sequences that are homologous to SEQ ID NO:2 are typically600 or more, 630 or more, or 660 or more amino acids. The amino acidsequence of the Marburg marburgvirus first consensus envelopeglycoprotein immunogen may be SEQ ID NO:4 (MARV RAV), a fragment of SEQID NO:4, an amino acid sequence that is homologous to SEQ ID NO:4, or afragment of an amino acid sequence that is homologous to SEQ ID NO:4.Amino acid sequences that are homologous to SEQ ID NO:4 are typically95% or more, 96% or more, 97% or more, 99% or more, or 99% or more,homologous to SEQ ID NO:4. Fragments of SEQ ID NO:4 or fragments ofamino acid sequences that are homologous to SEQ ID NO:4 are typically600 or more, 637 or more, or 670 or more amino acids. The amino acidsequence of the Marburg marburgvirus second consensus envelopeglycoprotein immunogen may be SEQ ID NO:5 (MARV OZO), a fragment of SEQID NO:5, an amino acid sequence that is homologous to SEQ ID NO:5, or afragment of an amino acid sequence that is homologous to SEQ ID NO:5.Amino acid sequences that are homologous to SEQ ID NO:5 are typically95% or more, 96% or more, 97% or more, 99% or more, or 99% or more,homologous to SEQ ID NO:4. Fragments of SEQ ID NO:5 or fragments ofamino acid sequences that are homologous to SEQ ID NO:5 are typically600 or more, 637 or more, or 670 or more amino acids. The amino acidsequence of the Marburg marburgvirus third consensus envelopeglycoprotein immunogen may be SEQ ID NO:6 (MARV MUS), a fragment of SEQID NO:6, an amino acid sequence that is homologous to SEQ ID NO:6, or afragment of an amino acid sequence that is homologous to SEQ ID NO:6.Amino acid sequences that are homologous to SEQ ID NO:6 are typically95% or more, 96% or more, 97% or more, 99% or more, or 99% or more,homologous to SEQ ID NO:6. Fragments of SEQ ID NO:6 or fragments ofamino acid sequences that are homologous to SEQ ID NO:6 are typically600 or more, 637 or more, or 670 or more amino acids. The amino acidsequence may optionally comprise a leader sequences such as the IgEleader. In some embodiments, the composition further comprises a nucleicacid sequence that encodes the Marburg marburgvirus Angola 2005 envelopeglycoprotein immunogen. The amino acid sequence of the Marburgmarburgvirus Angola 2005 envelope glycoprotein immunogen may be SEQ IDNO:3 (MARV ANG), a fragment of SEQ ID NO:3, an amino acid sequence thatis homologous to SEQ ID NO:3, or a fragment of an amino acid sequencethat is homologous to SEQ ID NO:3. Amino acid sequences that arehomologous to SEQ ID NO:3 are typically 95% or more, 96% or more, 97% ormore, 99% or more, or 99% or more, homologous to SEQ ID NO:3. Fragmentsof SEQ ID NO:3 or fragments of amino acid sequences that are homologousto SEQ ID NO:3 are typically 600 or more, 637 or more, or 670 or moreamino acids. The amino acid sequence may optionally comprise a leadersequences such as the IgE leader.

Also provided is a composition comprising a nucleic acid sequence thatencodes a consensus Zaire ebolavirus envelope glycoprotein immunogen,and a nucleic acid sequence that encodes a consensus Sudan ebolavirusenvelope glycoprotein immunogen. The amino acid sequence of theconsensus Zaire ebolavirus envelope glycoprotein immunogen may be SEQ IDNO:1 (ZEBOV CON), a fragment of SEQ ID NO:1, an amino acid sequence thatis homologous to SEQ ID NO:1, or a fragment of an amino acid sequencethat is homologous to SEQ ID NO:1. Amino acid sequences that arehomologous to SEQ ID NO:1 are typically 95% or more, 96% or more, 97% ormore, 99% or more, or 99% or more, homologous to SEQ ID NO:1. Fragmentsof SEQ ID NO:1 or fragments of amino acid sequences that are homologousto SEQ ID NO:1 are typically 600 or more, 630 or more, or 660 or moreamino acids. The amino acid sequence of the consensus Sudan ebolavirusenvelope glycoprotein immunogen may be SEQ ID NO:2 (SUDV CON), afragment of SEQ ID NO:2, an amino acid sequence that is homologous toSEQ ID NO:2, or a fragment of an amino acid sequence that is homologousto SEQ ID NO:2. Amino acid sequences that are homologous to SEQ ID NO:1are typically 95% or more, 96% or more, 97% or more, 99% or more, or 99%or more, homologous to SEQ ID NO:2. Fragments of SEQ ID NO:2 orfragments of amino acid sequences that are homologous to SEQ ID NO:2 aretypically 600 or more, 630 or more, or 660 or more amino acids. Theamino acid sequence may optionally comprise a leader sequences such asthe IgE leader.

A composition comprising a nucleic acid sequence that encodes aconsensus Zaire ebolavirus envelope glycoprotein immunogen, a nucleicacid sequence that encodes a consensus Sudan ebolavirus envelopeglycoprotein immunogen, and a nucleic acid sequence that encodes aMarburg marburgvirus Angola 2005 envelope glycoprotein immunogen isprovided. The nucleic acid sequence that encodes a consensus Zaireebolavirus envelope glycoprotein immunogen may be SEQ ID NO:64, afragment of SEQ ID NO:64, a nucleic acid sequence that is homologous toSEQ ID NO:64, or a fragment of a nucleotide sequence that is homologousto SEQ ID NO:64. Nucleic acid sequences that are homologous to SEQ IDNO:64 are typically 95% or more, 96% or more, 97% or more, 99% or more,or 99% or more, homologous to SEQ ID NO:64. Fragments of SEQ ID NO:64 orfragments of amino acid sequences that are homologous to SEQ ID NO:64typically encode 600 or more, 630 or more, or 660 or more amino acids ofthe consensus Zaire ebolavirus envelope glycoprotein immunogen encodedby SEQ ID NO:64. The nucleic acid sequence that encodes a consensusSudan ebolavirus envelope glycoprotein immunogen may be SEQ ID NO:65, afragment of SEQ ID NO:65, a nucleic acid sequence that is homologous toSEQ ID NO:65, or a fragment of a nucleotide sequence that is homologousto SEQ ID NO:65. Nucleic acid sequences that are homologous to SEQ IDNO:65 are typically 95% or more, 96% or more, 97% or more, 99% or more,or 99% or more, homologous to SEQ ID NO:65. Fragments of SEQ ID NO:65 orfragments of amino acid sequences that are homologous to SEQ ID NO:65typically encode 600 or more, 630 or more, or 660 or more amino acids ofthe consensus Sudan ebolavirus envelope glycoprotein immunogen encodedby SEQ ID NO:65. The nucleic acid sequence that encodes Marburgmarburgvirus Angola 2005 envelope glycoprotein immunogen may be SEQ IDNO:66, a fragment of SEQ ID NO:66, a nucleic acid sequence that ishomologous to SEQ ID NO:66, or a fragment of a nucleotide sequence thatis homologous to SEQ ID NO:66. Nucleic acid sequences that arehomologous to SEQ ID NO:66 are typically 95% or more, 96% or more, 97%or more, 99% or more, or 99% or more, homologous to SEQ ID NO:66.Fragments of SEQ ID NO:66 or fragments of amino acid sequences that arehomologous to SEQ ID NO:66 typically encode 600 or more, 630 or more, or670 or more amino acids of the Marburg marburgvirus Angola 2005 envelopeglycoprotein immunogen encoded by SEQ ID NO:66. The nucleic acidsequences may optionally include sequences that encode leader sequencessuch as the IgE leader linked to the sequences encoding the immunogens.

Each of the different nucleic acid sequences may be on a single nucleicacid molecule, may each be on a separate nucleic acid molecules orvarious permutations. Nucleic acid molecules may be plasmids.

The composition may be formulated for delivery to an individual usingelectroporation.

The composition may further comprise nucleic acid sequences that encodeone or more proteins selected from the group consisting of: IL-12, IL-15and IL-28.

The composition may be used in methods of inducing an immune responseagainst a filovirus. The filovirus may be selected from the groupconsisting of: Marburgvirus, Ebolavirus Sudan and Ebolavirus Zaire.

Methods of treating an individual who has been diagnosed with filoviruscomprising administering a therapeutically effective amount of thecomposition to an individual are provided. The filovirus may be selectedfrom the group consisting of: Marburgvirus, Ebolavirus Sudan andEbolavirus Zaire.

Method of preventing filovirus infection in an individual are provided.The methods comprise administering a prophylactically effective amountof the composition to an individual. The filovirus may be selected fromthe group consisting of: Marburgvirus, Ebolavirus Sudan and EbolavirusZaire.

Compositions comprising two or more proteins selected from the groupconsisting of: a consensus Zaire ebolavirus envelope glycoproteinimmunogen, a consensus Sudan ebolavirus envelope glycoprotein immunogen,a Marburg marburgvirus Angola 2005 envelope glycoprotein immunogen, afirst consensus Marburg marburgvirus envelope glycoprotein immunogen, asecond consensus Marburg marburgvirus envelope glycoprotein immunogenand a third consensus Marburg marburgvirus envelope glycoproteinimmunogen are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C refer to the polyvalent-vaccine construction strategy andexpression experiments in Example 1. FIG. 1A shows phylogenetic treesfor MGP (top), SGP (lower right), and ZGP (lower left). Significantsupport values are indicated (*) as verified by bootstrap analysis. Aconsensus strategy was adopted for the ZGP and SGP immunogens (CONVACCINE). Scale bars signify distance of amino acids per site andanalyses were conducted using MEGA version 5 software. GP transgeneswere commercially synthesized, genetically optimized, and subcloned intomodified pVAX1 mammalian expression vectors. Antigen expression wasanalyzed following transfection of HEK 293T cells by Westernimmunoblotting and FACS. Western immunoblotting results are shown inFIG. 1B and FACS in FIG. 1C. For a comparative control, rVSV expressingMGP, SGP, or ZGP was run concurrently with each GP sample andspecies-specific anti-GP1 mAbs were used for detection. Size isindicated (kDa). For FACS, transfected cells were indirectly stainedwith mouse-derived GP-specific serum reagents followed by extensivewashing and goat anti-mouse IgG and MHC class I. Western immunoblottingand FACS experiments were repeated at least three times with similarresults. Significance for unrooted phylogenetic trees was determined bymaximum-likelihood method and verified by bootstrap analysis andsignificant support values (≥80%; 1,000 bootstrap replicates) weredetermined by MEGA version 5 software.

FIGS. 2A-2H show results from experiments in Example 1 in which completeprotection against MARV and ZEBOV challenge was observed. Animalsurvival data is shown in FIG. 2A and FIG. 2E. FIG. 2A shows trivalentvaccinated animals survived post MARV challenge while control animalsall died by day 10. FIG. 2E shows trivalent vaccinated animals survivedpost ZEBOV challenge while control animals all died by day 7. Data for %change in body weight for vaccinated and control animal are displayed inFIG. 2B for vaccinated challenged with MARV. The y axis indicates changein body weight as shown in FIG. 2F. The light solid line is forTrivalent vaccinated animals. The light dashed line is for TriAVE, theaverage results of the Trivalent vaccinated animals. The dark solid lineis for control animals. The dark dashed line is for Control AVE, theaverage result for the control animals. The light solid lines and lightdashed lines remain steady on the graph in the days post challengeshowing no significant weight loss among vaccinated animals. The darksolid lines and dark dashed lines decline on the graph from days 0-9post challenge ending with the dagger denote animals that succumbed todisease by day 10. Data for % change in body weight for vaccinated andcontrol animal are displayed in FIG. 2F for vaccinated challenged withZEBOV. The y axis shows change in body weight as a percent. The lightsolid line is for Trivalent vaccinated animals. The light dashed line isfor TriAVE, the average results of the Trivalent vaccinated animals. Thedark solid line is for control animals. The dark dashed line is forControl AVE, the average result for the control animals. The light solidlines and light dashed lines remain steady on the graph in the days postchallenge showing no significant weight loss among vaccinated animals.The dark solid lines and dark dashed lines decline on the graph fromdays 0-6 post challenge ending with the dagger denote animals thatsuccumbed to disease before day 8. (n=3 for gpMARV and n=6 for gpZEBOV).Binding Abs (FIG. 2C and FIG. 2G) and NAbs (FIG. 2D and FIG. 2H) weremeasured in serum from vaccinated animals before (Pre) and after thefirst (1×) and second (2×) immunizations. Analysis was conducted onpooled serum (FIG. 2H). *p<0.1; ***p<0.001; ****p<0.0001.

FIGS. 3A-3C show results from Example 1 demonstrating induction ofneutralizing Abs. B cell responses were assessed in mice (n=5/group) 20days following each of two vaccinations, spaced three weeks betweeninjections with 40 μg of E-DNA vaccination. FIG. 3A shows serumGP-specific IgG responses from vaccinated (solid lines) mice or pre-bled(dotted lines) mice were measured by ELISA. The data is summarized inFIG. 3B. All responses from pEBOS- and pEBOZ-immunized animals weremeasured against sucrose-purified ZGP since SGP was not available forthis study. IgG responses from pMARV-immunized mice were measuredagainst MARV-Ozolin GP or with negative control sucrose-purified Nipah Gprotein, Neutralization activity of serum samples was measured againstZEBOV-EGFP, SUDV-Boniface and MARV-Angola in a BSL-4 facility and NAbtiters are shown in FIG. 3C. NAbs against SUDV-Boniface were assayedbased on cytopathic effect (CPE) on CV-1 cells and those againstMARV-Angola were assayed using an immunofluorescent assay. Averages areshown in FIG. 3B and FIG. 3C and error bars represent SEM. Groupanalyses were completed by matched, two-tailed, unpaired t test.Experiments were repeated at least two times with similar results and*p<0.1; **p<0.01;***p<0.001.

FIGS. 4A-4D shows refer to broad T cells responses generated byvaccination. In FIG. 4A H-2^(b) (light bars) and H-2^(d) (dark bars)mice (n=5/group) were immunized twice with either pMARV, pEBOS or pEBOZDNA, and IFNγ responses were measured by IFNγ ELISPOT assay. Splenocytesharvested 8 days after the second immunization were incubated in thepresence of individual GP peptides (15-mers overlapping by 9 aminoacids) and results are shown in stacked bar graphs. Epitope-containingpeptides were identified (≥10 AVE spots AND ≥80% response rate),confirmed by flow cytometry and characterized in the population of totalactivated IFNγ+ and CD44+ CD4+ and/or CD8+ T cells (Tables 1-6), andpeptide numbers of positive inducers are indicated above the bars.Peptides containing CD4+ epitopes alone, CD8+ epitopes alone (*), anddual CD4+ and CD8+ epitopes (**) are numbered. Putative shared and/orpartial epitopes were explored for contiguous positive peptide responses(Tables 1-6). FIG. 4B shows amino acid similarity plots comparing GPsequences from MARV, SUDV, and ZEBOV viruses displayed in FIG. 1A. FIG.4C is a diagram showing putative domains within the ZEBOV GP (GenBank#VGP_EBOZM). SP, signal peptide; RB, receptor binding; MUC, mucin-likeregion; FC, furin cleavage site; TM, transmembrane region. In FIG. 4D,total subdominant (darker shade) and immunodominant (lighter shade) Tcell epitopic responses are displayed as a percentage of the total IFNγresponse generated by each vaccine. Experiments were repeated at leasttwo times with similar results.

FIGS. 5A-5D show data from experiment assessing protective ‘single-dose’vaccination induced neutralizing Abs and CTL. H-2^(k) mice (n=10/group)were vaccinated once i.m. with pEBOZ E-DNA and then challenged 28 dayslater with 1,000 LD₅₀ of mZEBOV in a BSL-4 facility. Mice were weigheddaily and monitored for disease progression. Animal survival data inFIG. 5A. Vaccinated animals survived challenge while control animalsdied by day 7. FIG. 5B shows data for % change in body weight inchallenged animals. Data from immunized animals is shown as a solidlight line; the average data for immunized animals is shown as a dashedlight line. Data from control animals is shown as a solid dark line; theaverage data for control animals is shown as a dashed dark line. Thelight solid lines and light dashed lines remain steady within the rangeof about 85%-120% on the graph in the days post challenge showing nosignificant weight loss among vaccinated animals. The dark solid linesand dark dashed lines decline on the graph from days 0-6 post challengeending with the dagger denote animals that succumbed to disease by day7. NAbs measured prior to challenge; the data shown in FIG. 5C. T cellresponses after a single pEBOZ immunization as measured by FACS aresummarized as AVE % of total CD44+/IFNγ+ CD4+ (dark) or CD8+ (light)cells in FIG. 5D. T_(h)1-type effector markers were assessed (TNF andT-bet) and data for CD44+/IFNγ+ CD4+ and CD8+ T cells were compared withtotal T cell data which was as follows: For Total Cells: TNF 2.9±0.8,Tbet 13.0±1.1. For CD4+/CD44+/IFNγ+ Cells: TNF 61.4±3.1, Tbet 72.6±2.0.For CD8+/CD44+/IFNγ+ Cells: TNF 33.0±3.3, Tbet 992.1±1.4 (*p<0.1;***p<0.001; ****p<0.0001). Group analyses were completed by matched,two-tailed, unpaired t test and survival curves were analyzed bylog-rank (Mantel-Cox) test. Experiments were performed twice withsimilar results and error bars represent SEM.

FIG. 6 shows a GP-specific T cell gating disclosed in Example 1.

FIGS. 7A and 7B show that vaccination experiments in Example 1 generatedrobust T cells.

FIGS. 8A and 8B show T cell induction by ‘single-dose’ vaccinationdisclosed in Example 1.

FIG. 9 depicts the vaccination strategy against Ebola. Ebola viralglycoproteins are the major target for vaccines. Currently, threevaccines are currently in clinical trials which are immunogenicprotective in non-human primates (NHPs) and have single dose protection.However, these vaccines develop anti-vector immunity, show adversereactions in human clinical trials, have uncertain duration of memoryresponse and may not be suitable for all populations.

FIG. 10 refers to the EBOV glycoprotein vaccine construction andformulation strategy and expression experiments in Example 7.

FIG. 11 shows results from experiments in Example 7 demonstrating asingle immunization of DNA vaccine is immunogenic in mice.

FIG. 12 shows results from Example 7 demonstrating a single immunizationis fully protective in mice against lethal mouse-adapted Ebola viruschallenge.

FIG. 13 shows results from Example 7 demonstrating individual GP DNAvaccine constructs induce robust memory responses in mice.

FIG. 14 shows results from Example 7 demonstrating efficacy of GP DNAvaccines in non-human primates.

FIG. 15 shows results from Example 7 demonstrating GP DNA vaccineformulations are immunogenic in NHPs.

FIG. 16 shows results from Example 7 demonstrating GP DNA formulationvaccines protect against lethal Zaire Ebola virus (Makona) challenge.

FIG. 17 shows EBOV-001 phase I clinical (NCT02464670) study strategy.

FIG. 18 shows results from Example 7 demonstrating EBOV-001seroconversion in the phase I clinical trial.

FIG. 19 shows results from Example 7 demonstrating induction of Ebola GPSpecific T-Cell Responses (ELISpot) in Representative Patients.

FIG. 20 shows results from Example 7 demonstrating a comparison withother Ebola vaccine platforms currently in clinical trials.

FIG. 21 shows the cohort descriptions and cohort demographics for theELISA experiments from Example 8.

FIG. 22 shows results from Example 8 demonstrating ELISA Titers byCohort and Time point for cohorts 1-3.

FIG. 23 shows results from Example 8 demonstrating ELISA Titers byCohort and Time point for cohorts 4 and 5.

FIG. 24 shows results from Example 8 demonstrating an ELISA Summary forall cohorts.

FIG. 25 shows the cohort descriptions and cohort demographics for theELISpot experiments from Example 8.

FIG. 26 shows results from Example 8 demonstrating subject responses bypeptide pool for cohort 1.

FIG. 27 shows results from Example 8 demonstrating subject responses bypeptide pool for cohort 2.

FIG. 28 shows results from Example 8 demonstrating subject responses bypeptide pool for cohort 3.

FIG. 29 shows results from Example 8 demonstrating subject responses bypeptide pool for cohort 4.

FIG. 30 shows results from Example 8 demonstrating subject responses bypeptide pool for cohort 5.

FIG. 31 shows results from Example 8 demonstrating ELISpot summary bycohort.

FIG. 32 shows results from Example 8 demonstrating ELISpot summary bycohort.

FIG. 33 shows results from Example 8 demonstrating analysis of vaccineresponders for all subjects.

FIG. 34 shows results from Example 8 demonstrating analysis of vaccineresponders for subjects with baseline outliers removed.

FIG. 35 shows the cohort descriptions and cohort demographics for theICS experiments from Example 8.

FIG. 36 shows results from Example 8 demonstrating Wilcoxon Pairedanalysis based on cohort for ICS experiments.

FIG. 37 shows results from Example 8 demonstrating ICS analysis forcohort 3 (INO4201 ID) Cytokines in CD4+ T cells.

FIG. 38 shows results from Example 8 demonstrating ICS analysis forcohort 3 Cytokines in CD8+ T cells.

FIG. 39 shows results from Example 8 demonstrating a detailed analysisof vaccine responders for each subject in cohorts 1-3.

FIG. 40 shows results from Example 8 demonstrating a detailed analysisof vaccine responders for each subject in cohorts 4-5.

FIG. 41 shows results from Example 8 demonstrating median responses bycohort and pool.

FIG. 42 shows results from Example 8 demonstrating mean responses bycohort and pool.

FIG. 43 shows results demonstrating that bivalent and trivalent DNAvaccines elicit long-term immune responses in vivo in cynomolgusmacaques. Serum and peripheral blood mononuclear cells (PBMCs) werecollected 2-weeks post-each DNA injection and monthly following finalinjection. Total IgG endpoint titers against Guinea-GP and Mayinga-GPwere assayed by ELISA. Cellular immune responses against Ebola GPfollowing DNA immunization. PMBCs were collected from NHPs and assayedby ELISPOT-IFNγ for T cells responses following stimulation with poolsof GP peptides.

FIG. 44, comprising FIG. 44A through FIG. 44C, shows resultsdemonstrating protection in cynomolgous macaques with bivalent andtrivalent DNA vaccines against lethal EBOV challenge. FIG. 44A)Survival. Animals were challenged with 1000TCID50 of lethal GuineaMakona C07 EBOV and survival was monitored for 28 days post-challenge,FIG. 44B) Clinical score. Clinical signs of disease were monitoredthroughout the course of infection. FIG. 44C) Viral load. Viremia duringthe course of infection was assayed from blood by TCID50 assay.

FIG. 45 shows a quantification ELISA using the ADI human anti-ZaireEbola virus glycoprotein IgG kit to assess the amount of ZEBOV specificIgG antibody in the sera of each vaccinated subject at study entry and 2weeks post each immunization. Statistical analyses were performed usingtwo tailed Wilcoxon Sign Rank test.

FIG. 46 shows an Interferon Gamma ELISpot response. PBMCs were isolatedfrom vaccinated subjects at study entry and 2 weeks post eachimmunization and stimulated with Ebola peptides spanning the full lengthGP in an IFNg ELISpot assay. The graph represents EBOV-GP specific spotforming units per million PBMCs. Lines within boxes represent Medianresponse. Individual dots represent outlier data.

FIG. 47 depicts experimental results demonstrating long-termimmunogenicity with Ebola GP DNA vaccine formulations in non-humanprimates (NHPs). Strong antibody responses are observed greater than 6months post-vaccination.

FIG. 48 depicts the Memory study of intramuscular delivery of EBOV GPDNA vaccine. NHPs were immunized over a 3 month period with bivalent ortrivalent EBOV GP DNA vaccine formulations. The immune responses werefollowed over a 12 months following the final dose. A 1 year boost wasgiven at month 13.

FIG. 49 depicts the total IgG endpoint titers of animals receiving atrivalent DNA GP formulation. An increase in total IgG antibody responsewas observed following the 1-year boost.

FIG. 50 depicts the total IgG endpoint titers of animals receiving abivalent or trivalent DNA GP formulation. An increase in total IgGantibody response was observed following the 1-year boost.

FIG. 51 depicts the ELISPOT of trivalent DNA GP formulations. IncreaseIFNγ ELISPOT responses following the 1 year boost. Magnitude of boost isnot as high in the 3 injection group.

FIG. 52 depicts the ELISPOT of bivalent or trivalent DNA GPformulations. Increase IFNγ ELISPOT responses following the 1 yearboost. There was a remarkably strong boost in single immunization group.

FIG. 53 depicts a summary of IFNγ ELISPOT results. 17/19 animals hadincreased T cell responses following the boost. 1/19 animals maintainedthe same level of T cell responses. 1/19 animals had worse T cellresponses following the boost, however this animal was consistently highover the past year in the 3 injection group.

FIG. 54 depicts an exemplary IFNγ ELISPOT for animals in the singleimmunization group at the 12 month time point, before the 1-year boost.

FIG. 55 depicts an exemplary IFNγ ELISPOT for animals in the singleimmunization group after the 1-year boost.

FIG. 56 depicts the results of the IM memory study. The EBOV GP DNAvaccines elicit long term immune response with a strong recall followinga 1 year boost. Intradermal delivery studies are carried out to studythe immune response of intradermal EBOV GP DNA vaccines.

FIG. 57 depicts a summary of the EBOV DNA vaccine clinical trial(EBOV-001). Healthy volunteers receive a 3 dose regimen of INO-4201,INO-4202, INO-4212 or INO-4212 and INO-9021.

FIG. 58 depicts a comparison of binding antibodies in EBOV-001 to rVSVEBOV. All EBOV-001 cohorts had significant increases at both weeks 6 and14 compared to week 0.

FIG. 59 depicts a summary of the Intradermal Delivery of EBOV GP DNAvaccine study. Cohorts were added to explore dosing, dose regimens anduse of IL-12 DNA as an immuno-adjuvant.

FIG. 60 depicts the results of ID cohorts at week 14. Sereoreactivitywas observed in 125/127 (98.4%) subjects in all ID cohorts.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In one aspect of the invention, it is desired that the consensus antigenprovides for improved transcription and translation, including havingone or more of the following: low GC content leader sequence to increasetranscription; mRNA stability and codon optimization; eliminating to theextent possible cis-acting sequence motifs (i.e., internal TATA-boxes).

In some aspects of the invention, it is desired to generate a consensusantigen that generates a broad immune response across multiple strains,including having one or more of the following: incorporate all availablefull-length sequences; computer generate sequences that utilize the mostcommonly occurring amino acid at each position; and increasecross-reactivity between strains.

Diversity among the Filoviridae is relatively high. Intensive effortshave been aimed at developing a universal and broadly-reactive filovirusvaccine that would ideally provide protection against multiple speciesresponsible for the highest human case-fatality rates. However, thisproves difficult due to the relative high level of diversity among theFilovirida. The EBOV are currently classified into five distinctspecies, Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SUDV), Restonebolavirus (RESTV), Bundibugyo ebolavirus (BDBV) and Taï Forestebolavirus (TAFV; formerly Cote d'Ivoire ebolavirus), the first tworesponsible for the highest lethality rates and the most likelycandidates for weaponization. Diversity is lower among the Marburgviruses (MARV) of which can also be up to 90% lethal. Currently, thereis only one classified species, Marburg marburgvirus (formerly LakeVictoria marburgvirus), although a recent amendment proposes that itcontain two viruses including the Ravn virus (RAVV). Adding to thecomplexity for polyvalent-vaccine development, the MARV and EBOV arehighly divergent, in which there exists about 67% divergence at thenucleotide level. Furthermore, phylogenetic diversity among thefiloviral GP is also very high (82% overall). These allude to thepotential of the filoviruses to evolve, as demonstrated by the recentemergence of BDBV in 2007. Therefore, due to relative divergence amongthe Filoviridae, we hypothesized that development of an effectivepolyvalent-filovirus vaccine will likely require a cocktail ofimmunogenic components.

A synthetic polyvalent-filovirus DNA vaccine against Marburgmarburgvirus (MARV), Zaire ebolavirus (ZEBOV), and Sudan ebolavirus(SUDV) was developed. The novel polyvalent-filovirus vaccine comprisedby three DNA plasmids encoding the envelope glycoprotein (GP) genes ofMarburg marburgvirus (MARV), Sudan ebolavirus (SUDV) or Zaire ebolavirus(ZEBOV), adopting the multiagent approach. As a filoviral vaccinecandidate, an enhanced DNA (DNA)-based platform exhibits many advantagesgiven recent advances in genetic optimization and delivery techniques(Bagarazzi M L, et al. (2012). Immunotherapy Against HPV16/18 GeneratesPotent TH1 and Cytotoxic Cellular Immune Responses. Sci Transl Med 4:155ra138; Kee S T, Gehl J, W. L E (2011). Clinical Aspects ofElectroporation, Springer, New York, N.Y.; Hirao L A, et al. (2011).Multivalent smallpox DNA vaccine delivered by intradermalelectroporation drives protective immunity in nonhuman primates againstlethal monkeypox challenge. J Infect Dis 203: 95-102). As such, each GPwas genetically-optimized, subcloned into modified mammalian expressionvectors, and then delivered using in vivo electroporation (EP).

Preclinical efficacy studies were performed in guinea pigs and miceusing rodent-adapted viruses, while murine T cell responses wereextensively analyzed using a novel modified assay described herein. Tcell responses were extensively analyzed including the use of a novelmethod for epitope identification and characterization described herein.This model provides an important preclinical tool for studyingprotective immune correlates that could be applied to existingplatforms.

Vaccination in preclinical rodent studies was highly potent, elicitedrobust neutralizing antibodies (NAbs) and CTL expressing T_(h)1-typemarkers, and completely protected against MARV and ZEBOV challenge.Comprehensive T cell analysis as extensively analyzed using a novelmodified assay described herein (Shedlock D J, et al. (2012).Vaccination with synthetic constructs expressing cytomegalovirusimmunogens is highly T cell immunogenic in mice. Hum Vaccin Immunother8: 1668-1681) revealed cytotoxic T lymphocytes of great magnitude,epitopic breadth, and T_(h)1-type marker expression. In total, 52 novelT cell epitopes from two different mouse genetic backgrounds wereidentified (19 of 20 MARV epitopes, 15 of 16 SUDV, and 18 of 22 ZEBOV)and occurred primarily in highly conserved regions of their respectiveglycoproteins (GPs). These data represent the most comprehensive reportof preclinical glycoprotein epitopes to date.

In developing a strategy to provide protection against multiple speciesresponsible for the highest human case-fatality rates, we focused onMARV, SUDV, and ZEBOV. Due to their relative divergence, we hypothesizedthat development of a polyvalent-filovirus vaccine would require acocktail of components that can be quickly and easily adapted inresponse to future outbreak strains and/or species. While overalldiversity among the EBOV is about 33%, amino acid identity increasessubstantially when SUDV and ZEBOV are analyzed separately (-94% identitywithin each species). Therefore, as shown in FIG. 1A, a two componentstrategy for coverage of the most lethal EBOV, one plasmid GP vaccinefor SUDV and another for ZEBOV was designed. Since GP diversity amongeach species was relatively low (5.6% for SUDV and 7.1% for ZEBOV),consensus immunogens were developed increase inter-species coverage, astrategy shown previously to enhance protection among divergent strainsof influenza and HIV. These GP sequences were consensus for all reportedoutbreak sequences (GenBank) as determined by alignment using Vector NTIsoftware (Invitrogen, CA, USA). Non-consensus residues, 4 amino acidseach in SUDV (95, 203, 261, and 472) and ZEBOV (314, 377, 430, and 440),were weighted towards Gulu and Mbomo/Mbanza, respectively. Gulu waschosen since it was responsible for the highest human case-fatality rateof any Filoviridae outbreak (n=425), while Mbomo/Mbanza was chosen sincethey were the most recent and lethal outbreaks with published sequencedata. The consensus GP for SUDV (SUDV CON VACCINE) and ZEBOV (ZEBOV CONVACCINE) were phylogenetically intermediary their parentally alignedstrains.

Identification of proteins in FIG. 1A are as follows: MARV Durba(05DRC99) '99: ABE27085; Uganda (01Uga07) '07: ACT79229; Durba (07DRC99)'99: ABE27078; Ozolin '75: VGP_MABVO; Musoke '80: VGP_MABVM; Popp '67:VGP_MABVP; Leiden '08: AEW11937; Angola '05: VGP_MABVA; Ravn '87:VGP_MABVR; Durba (09DRC99) '99; ABE27092; Uganda (02Uga07) '07:ACT79201. SUDV: Boniface '76: VGP_EBOSB; Maleo '79: VGP_EBOSM; Yambio'04: ABY75325; Gulu '00: VGP_EBOSU. ZEBOV: Booue '96: AAL25818; Mayibout'96: AEK25495; Mekouka '94: AAC57989, VGP_EBOG4; Kikwit '95: VGP_EBOZ5;Yambuku (Ekron) '76: VGP_EBOEC; Yambuku (Mayinga) '76: VGP_EBOZM; Kasai'08: AER59712; Kassai '07: AER59718; Etoumbi '05: ABW34742;Mbomo/Mbandza '03: ABW34743.

A sequence listing provided herewith contains a list of 72 sequencesincluding the following

SEQ ID NO:1 is the amino acid sequence of ZEBOV CON (CONGP1), which is aconsensus Zaire ebolavirus envelope glycoprotein immunogen.

SEQ ID NO:2 is the amino acid sequence of SUDV CON, which is a consensusSudan ebolavirus envelope glycoprotein immunogen.

SEQ ID NO:3 is the amino acid sequence of MARV or MARV ANG, which theamino acid sequence of the Marburg marburgvirus Angola 2005 envelopeglycoprotein and a Marburg marburgvirus Angola 2005 envelopeglycoprotein immunogen.

SEQ ID NO:4 is the amino acid sequence of MARV CON1, which is the firstconsensus Marburg marburgvirus envelope glycoprotein immunogen.

SEQ ID NO:5 is the amino acid sequence of MARV CON2, which is the secondconsensus Marburg marburgvirus envelope glycoprotein immunogen.

SEQ ID NO:6 is the amino acid sequence of MARV CON3, which is the thirdconsensus Marburg marburgvirus envelope glycoprotein immunogen.

SEQ ID NOs:7-25 are peptides derived from MARV ANG.

SEQ ID NO:26-41 are peptides derived from SUDV CON.

SEQ ID NO: 42-62 are peptides derived from ZEBOV CON.

SEQ ID NO:63 is the sequence of the IgE signal peptide:MDWTWILFLVAAATRVHS.

SEQ ID NO:64 is the nucleotide sequence insert in plasmid pEBOZ whichencodes consensus Zaire ebolavirus envelope glycoprotein immunogen.

SEQ ID NO:65 is the nucleotide sequence insert in plasmid pEBOS whichencodes consensus Sudan ebolavirus envelope glycoprotein immunogen.

SEQ ID NO:66 is the nucleotide sequence insert in plasmid pMARZ ANGwhich encodes the Marburg marburgvirus Angola 2005 envelopeglycoprotein.

SEQ ID NO:67 is the amino acid sequence of ZEBOVGUI (GuineaGP), which isa consensus Zaire ebolavirus envelope glycoprotein immunogen isolatedfrom the 2014 Outbreak in Guinea.

SEQ ID NO:68 is the amino acid sequence of ZEBOVCON2 (CONGP2), which isa second consensus Zaire ebolavirus envelope glycoprotein.

SEQ ID NO:69 is the nucleotide sequence insert in plasmid pZEBOVGUIwhich encodes consensus Zaire ebolavirus envelope glycoprotein immunogenisolated from the 2014 Outbreak in Guinea.

SEQ ID NO:70 is the nucleotide sequence insert in plasmid pEBOZCON2which encodes a second consensus Zaire ebolavirus envelope glycoprotein.

SEQ ID NO:71 is the nucleotide sequence of plasmid pEBOZCON2 whichencodes a second consensus Zaire ebolavirus envelope glycoprotein.

SEQ ID NO:72 is the nucleotide sequence insert in plasmid pZEBOVGUIwhich encodes a Zaire ebolavirus envelope glycoprotein immunogenisolated from the 2014 Outbreak in Guinea.

In some embodiments, the strategy employs coding sequences for threefilovirus immunogens selected from: MARV, SUDV, ZEBOV, ZEBOVGUI andZEBOVCON2. MARV immunogen is the glycoprotein of the Angola 2005isolate. For SUDV ZEBOV, ZEBOVGUI, and ZEBOVCON2 concensus glycoproteinsequences were designed.

In some embodiments, the strategy employs coding sequences for fivefilovirus immunogens. Three MARV immunogens are provided. Consensusglycoprotein Ozolin, Musoke, or Ravn derived from three clusters, weredesigned. These three MARV immunogens are targets for immune responsestogether the SUDV, ZEBOV or ZEBOVCON2, ZEBOVGUI consensus glycoproteinsequences that were designed.

In some embodiments, the strategy employs coding sequences for sixfilovirus immunogens. Four MARV immunogens are provided: three consensusglycoproteins derived from three clusters were designed. These threeMARV immunogens are targets for immune responses together the SUDV,ZEBOV and ZEBOVCON2, ZEBOVGUI consensus glycoprotein sequences that weredesigned, the MARV immunogen is the glycoprotein of the Angola 2005isolate.

As a candidate for filoviral vaccines, DNA vaccines exhibit a multitudeof advantages including rapid and inexpensive up-scale production,stability at room temperature, and ease of transport, all of whichfurther enhance this platform from an economic and geographicperspective. Due to the synthetic nature of the plasmids, Ag sequencescan be quickly and easily modified in response to newly emergent speciesand/or expanded to include additional vaccine components and/or regimenfor rapid response during outbreak settings. For example, the MARVstrategies herein can be easily expanded for greater coverage by theco-administration of additional plasmids encoding consensus MARV GP(MGP) immunogens for other phylogenetic clusters.

While ‘first-generation’ DNA vaccines were poorly immunogenic, recenttechnological advances have dramatically improved their immunogenicityin clinical trials. Optimization of plasmid DNA vectors and theirencoded Ag genes have led to increases in in vivo immunogenicity.Cellular uptake and subsequent Ag expression are substantially amplifiedwhen highly-concentrated plasmid vaccine formulations are administeredwith in vivo electroporation, a technology that uses brief square-waveelectric pulses within the vaccination site to drive plasmids intotransiently permeabilized cells. In theory, a cocktail of DNA plasmidscould be assembled for directing a highly-specialized immune responseagainst any number of variable Ags. Immunity can be further directed byco-delivery with plasmid molecular adjuvants encoding species-specificcytokine genes as well as ‘consensus-engineering’ of the Ag amino acidsequences to help bias vaccine-induced immunity towards particularstrains. This strategy has been shown to enhance protection amongdivergent strains of influenza virus and HIV. Due in parts to thesetechnological advancements, immunization regimens including these DNAvaccines are highly versatile and extremely customizable.

1. Definitions.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used in thespecification and the appended claims, the singular forms “a,” “an” and“the” include plural referents unless the context clearly dictatesotherwise.

For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range of 6-9, the numbers 7 and 8 are contemplatedin addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6,9, and 7.0 are explicitlycontemplated.

a. Adjuvant

“Adjuvant” as used herein may mean any molecule added to the DNA plasmidvaccines described herein to enhance antigenicity of the one or moreconsensus filovirus immunogens encoded by the DNA plasmids and encodingnucleic acid sequences described hereinafter.

b. Antibody

“Antibody” may mean an antibody of classes IgG, IgM, IgA, IgD or IgE, orfragments, fragments or derivatives thereof, including Fab, F(ab′)2, Fd,and single chain antibodies, diabodies, bispecific antibodies,bifunctional antibodies and derivatives thereof. The antibody may be anantibody isolated from the serum sample of mammal, a polyclonalantibody, affinity purified antibody, or mixtures thereof which exhibitssufficient binding specificity to a desired epitope or a sequencederived therefrom.

c. Coding Sequence

“Coding sequence” or “encoding nucleic acid” as used herein may meanrefers to the nucleic acid (RNA or DNA molecule) that comprise anucleotide sequence which encodes a protein. The coding sequence mayalso comprise a DNA sequence which encodes an RNA sequence. The codingsequence may further include initiation and termination signals operablylinked to regulatory elements including a promoter and polyadenylationsignal capable of directing expression in the cells of an individual ormammal to whom the nucleic acid is administered. In some embodiments,the coding sequence may optionally further comprise a start codon thatencodes an N terminal methionine or a signal peptide such as an IgE orIgG signal peptide.

d. Genetic Construct

Genetic construct” as used herein refers to the DNA or RNA moleculesthat comprise a nucleotide sequence which encodes a protein, such as animmunogen. The genetic construct may also refer to a DNA molecule whichtranscribes RNA. The coding sequence includes initiation and terminationsignals operably linked to regulatory elements including a promoter andpolyadenylation signal capable of directing expression in the cells ofthe individual to whom the nucleic acid molecule is administered. Asused herein, the term “expressible form” refers to gene constructs thatcontain the necessary regulatory elements operable linked to a codingsequence that encodes a protein such that when present in the cell ofthe individual, the coding sequence will be expressed.

e. Complement

“Complement” or “complementary” as used herein may mean a nucleic acidmay mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairingbetween nucleotides or nucleotide analogs of nucleic acid molecules.

f. Consensus or Consensus Sequence

“Consensus” or “consensus sequence” as used herein may mean a syntheticnucleic acid sequence, or corresponding polypeptide sequence,constructed based on analysis of an alignment of multiple subtypes of aparticular filovirus antigen, that can be used to induce broad immunityagainst multiple subtypes or serotypes of a particular filovirusantigen.

Consensus Zaire ebolavirus envelope glycoprotein immunogen refers to SEQID NO:1, fragments of SEQ ID NO:1, variants of SEQ ID NO:1 and fragmentof variants of SEQ ID NO:1. (ZEBOV or ZEBOV CON or ZEBOV CON VACCINE).Plasmids comprising coding sequences of SEQ ID NO:1 may be referred toas pZEBOV or pEBOZ. Coding sequences for consensus Zaire ebolavirusenvelope glycoprotein immunogen include SEQ ID NO:64, fragments of SEQID NO:64, variants of SEQ ID NO:64 and fragment of variants of SEQ IDNO:64. Plasmid pEBOZ comprises SEQ ID NO:64.

Consensus Sudan ebolavirus envelope glycoprotein immunogen refers to SEQID NO:2, fragments of SEQ ID NO:2, variants of SEQ ID NO:2 and fragmentof variants of SEQ ID NO:2. (SUDV or SUDV CON or SUDV CON VACCINE)Plasmids comprising coding sequences of SEQ ID NO:2 may be referred toas pSUDV or pEBOS. Coding sequences for consensus Sudan ebolavirusenvelope glycoprotein immunogen include SEQ ID NO:65, fragments of SEQID NO:65, variants of SEQ ID NO:65 and fragment of variants of SEQ IDNO:65. Plasmid pEBOS comprises SEQ ID NO:65.

Marburg marburgvirus Angola 2005 envelope glycoprotein is not aconsensus but a protein sequence derived from an isolate. It hassequence SEQ ID NO:3. Marburg marburgvirus Angola 2005 envelopeglycoprotein immunogen refers to SEQ ID NO:3, fragments of SEQ ID NO:3,variants of SEQ ID NO:3 and fragment of variants of SEQ ID NO:3. (MARVor MARV ANG or MARV ANG or MARV ANG VACCINE) Plasmids comprising codingsequences of SEQ ID NO:3 may be referred to as pMARV or pMARV-ANG.Coding sequences for Marburg marburgvirus Angola 2005 envelopeglycoprotein immunogen include SEQ ID NO:66, fragments of SEQ ID NO:66,variants of SEQ ID NO:66 and fragment of variants of SEQ ID NO:66.Plasmid pMARV ANG comprises SEQ ID NO:66.

The first consensus Marburg marburgvirus envelope glycoprotein immunogenrefers to SEQ ID NO:4, fragments of SEQ ID NO:4, variants of SEQ ID NO:4and fragment of variants of SEQ ID NO:4. SEQ ID NO:4 is a Marburgmarburgvirus consensus sequence from the Ravn cluster consensus (Ravn,Durba (09DRC99) and Uganda (02Uga07Y). (MARV CON1 or MARV-RAV CON orMARV-RAV CON VACCINE) Plasmids comprising coding sequences of SEQ IDNO:4 may be referred to as pMARV-RAV.

The second consensus Marburg marburgvirus envelope glycoproteinimmunogen refers to SEQ ID NO:5, fragments of SEQ ID NO:5, variants ofSEQ ID NO:5 and fragment of variants of SEQ ID NO:5. SEQ ID NO:5 is aMarburg marburgvirus consensus sequence from the Ozolin clusterconsensus (Ozolin, Uganda (01Uga07), and Durba (05 and 07DRC99)). (MARVCON2 or MARV-OZO CON or MARV-OZO CON VACCINE) Plasmids comprising codingsequences of SEQ ID NO:5 may be referred to as pMARV-OZO.

The third consensus Marburg marburgvirus envelope glycoprotein immunogenrefers to SEQ ID NO:6, fragments of SEQ ID NO:6, variants of SEQ ID NO:6and fragment of variants of SEQ ID NO:6. SEQ ID NO:6 is a Marburgmarburgvirus consensus sequence from the Musoke cluster consensus(Musoke, Popp, and Leiden). (MARV CON1 or MARV-MUS CON or MARV-MUS CONVACCINE) Plasmids comprising coding sequences of SEQ ID NO:6 may bereferred to as pMARV-MUS.

Consensus Zaire ebolavirus GP envelope glycoprotein immunogen refers toSEQ ID NO:67, fragments of SEQ ID NO:67, variants of SEQ ID NO:67 andfragment of variants of SEQ ID NO:67. (ZEBOVGUI or ZEBOVGUI VACCINE).Plasmids comprising coding sequences of SEQ ID NO:67 may be referred toas pZEBOVGUI or pEBOZGUI. Coding sequences for consensus Zaireebolavirus Guinea 2014 envelope glycoprotein immunogen include SEQ IDNO:69, fragments of SEQ ID NO:69, variants of SEQ ID NO:69 and fragmentof variants of SEQ ID NO:69. Plasmid pEBOZGUI comprises SEQ ID NO:69.

A second consensus Zaire ebolavirus envelope glycoprotein immunogenrefers to SEQ ID NO:68 fragments of SEQ ID NO:68, variants of SEQ IDNO:68 and fragment of variants of SEQ ID NO:68. (ZEBOV2 or ZEBOVCON2 orZEBOVCON2 VACCINE) Plasmids comprising coding sequences of SEQ ID NO: 68may be referred to as pEBOVCON2. Coding sequences for second consensusZaire ebolavirus envelope glycoprotein immunogen include SEQ ID NO:70,fragments of SEQ ID NO:70, variants of SEQ ID NO:70 and fragment ofvariants of SEQ ID NO:70. Plasmid pEBOVCON2 comprises SEQ ID NO:70.

g. Constant Current

“Constant current” as used herein to define a current that is receivedor experienced by a tissue, or cells defining said tissue, over theduration of an electrical pulse delivered to same tissue. The electricalpulse is delivered from the electroporation devices described herein.This current remains at a constant amperage in said tissue over the lifeof an electrical pulse because the electroporation device providedherein has a feedback element, preferably having instantaneous feedback.The feedback element can measure the resistance of the tissue (or cells)throughout the duration of the pulse and cause the electroporationdevice to alter its electrical energy output (e.g., increase voltage) socurrent in same tissue remains constant throughout the electrical pulse(on the order of microseconds), and from pulse to pulse. In someembodiments, the feedback element comprises a controller.

h. Current Feedback or Feedback

“Current feedback” or “feedback” as used herein may be usedinterchangeably and may mean the active response of the providedelectroporation devices, which comprises measuring the current in tissuebetween electrodes and altering the energy output delivered by the EPdevice accordingly in order to maintain the current at a constant level.This constant level is preset by a user prior to initiation of a pulsesequence or electrical treatment. The feedback may be accomplished bythe electroporation component, e.g., controller, of the electroporationdevice, as the electrical circuit therein is able to continuouslymonitor the current in tissue between electrodes and compare thatmonitored current (or current within tissue) to a preset current andcontinuously make energy-output adjustments to maintain the monitoredcurrent at preset levels. The feedback loop may be instantaneous as itis an analog closed-loop feedback.

i. Decentralized Current

“Decentralized current” as used herein may mean the pattern ofelectrical currents delivered from the various needle electrode arraysof the electroporation devices described herein, wherein the patternsminimize, or preferably eliminate, the occurrence of electroporationrelated heat stress on any area of tissue being electroporated.

j. Electroporation

“Electroporation,” “electro-permeabilization,” or “electro-kineticenhancement” (“EP”) as used interchangeably herein may refer to the useof a transmembrane electric field pulse to induce microscopic pathways(pores) in a bio-membrane; their presence allows biomolecules such asplasmids, oligonucleotides, siRNA, drugs, ions, and water to pass fromone side of the cellular membrane to the other.

k. Feedback Mechanism

“Feedback mechanism” as used herein may refer to a process performed byeither software or hardware (or firmware), which process receives andcompares the impedance of the desired tissue (before, during, and/orafter the delivery of pulse of energy) with a present value, preferablycurrent, and adjusts the pulse of energy delivered to achieve the presetvalue. A feedback mechanism may be performed by an analog closed loopcircuit.

l. Fragment

“Fragment” may mean a polypeptide fragment of a filovirus immunogen thatis capable of eliciting an immune response in a mammal against filovirusby recognizing the particular filovirus antigen. The filovirus envelopeglycoprotein immunogen may optionally include a signal peptides and/or amethionine at position 1, proteins 98% or more homologous to theconsensus sequences set forth herein, proteins 99% or more homologous tothe consensus sequences set forth herein, and proteins 100% identical tothe consensus sequences set forth herein, in each case with or withoutsignal peptides and/or a methionine at position 1. A fragment may or maynot for example comprise a fragment of a filovirus immunogen linked to asignal peptide such as an immunoglobulin signal peptide for example IgEsignal peptide or IgG signal peptide.

Fragments of any of ZEBOV CON, SUDV CON, MARV ANG, MARV-RAV CON,MARV-OZO CON, MARV-MUS CON, ZEBOVGUI or ZEBOVCON2 or variants thereof,in each case with or without signal peptides and/or a methionine atposition 1, may comprise 20% or more, 25% or more, 30% or more, 35% ormore, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more,65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% ormore, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more,96% or more, 97% or more, 98% or more, 99% or more percent of the lengthof the particular full length ZEBOV CON, SUDV CON, MARV ANG, MARV-RAVCON, MARV-OZO CON, MARV-MUS CON, ZEBOVGUI or ZEBOVCON2 or variantsthereof. Fragments refer to fragments polypeptides 100% identical to thesequences ZEBOV CON, SUDV CON, MARV ANG, MARV-RAV CON, MARV-OZO CON,MARV-MUS CON, ZEBOVGUI or ZEBOVCON2 in each case with or without signalpeptides and/or a methionine at position 1. Fragments also refer tofragments of variants, i.e. polypeptides that 95% or more, 98% or more,or 99% or more homologous to the sequences ZEBOV CON, SUDV CON, MARVANG, MARV-RAV CON, MARV-OZO CON, MARV-MUS CON, ZEBOVGUI or ZEBOVCON2 ,in each case with or without signal peptides and/or a methionine atposition 1. The fragment may comprise a fragment of a polypeptide thatis 98% or more homologous, 99% or more homologous, or 100% identical tothe Filovirus immunogens set forth in SEQ ID NOs: 1-6, 67-68 andadditionally comprise a signal peptide such as an immunoglobulin signalpeptide which is not included when calculating percent homology. In someembodiments, a fragment of SEQ ID NOs: 1-6, 67-68 linked to a signalpeptide such as an immunoglobulin signal peptide for example IgE signalpeptide or IgG signal peptide. The fragment may comprise fragments ofSEQ ID NOs: 1-6, 67-68 including the N terminal methionine. Fragmentsalso refer to fragments of a polypeptide that is 95% or more, 98% ormore, or 99% or more homologous to the sequence disclosed in SEQ ID NOs:1-6, 67-68. If a signal peptide is present it is not included whencalculating percent homology.

In some embodiments, fragments of SEQ ID NOs: 1-6, 67-68 or variantsthereof may comprise 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 610, 620, 630, 640, 650, 660, 670 ormore contiguous amino acids of any of SEQ ID NOs:1-6 or variantsthereof. In some embodiments, fragments of SEQ ID NOs:1-6 or variantsthereof may comprise 15, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 610, 620, 630, 640, 650, 660, 670, 675 orless contiguous amino acids of any of SEQ ID NOs: 1-6, 67-68 or variantsthereof.

“Fragment” may also mean a fragment of a nucleic acid sequence thatencodes a filovirus immunogen, the nucleic acid fragment encoding afragment of filovirus immunogen that is capable of eliciting an immuneresponse in a mammal against filovirus by recognizing the particularfilovirus antigen. Fragments of nucleic acid fragment encoding afilovirus immunogen or variants thereof, in each case with or withoutsignal peptides and/or a methionine at position 1, may comprise 20% ormore, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more,50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% ormore, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more,93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% ormore, 99% or more percent of the length of the particular full lengthnucleic acid sequence that encodes a filovirus immunogen or variantsthereof. The fragment may comprise a fragment of nucleotide sequencethat encodes polypeptide that is 98% or more homologous, 99% or morehomologous, or 100% identical to the filovirus immunogens set forth inSEQ ID NOs: 1-6, 67-68 and additionally comprise a signal peptide suchas an immunoglobulin signal peptide which is not included whencalculating percent homology. In some embodiments, fragment ofnucleotide sequence that encodes a fragment of SEQ ID NOs: 1-6, 67-68linked to a signal peptide such as an immunoglobulin signal peptide forexample IgE signal peptide or IgG signal peptide. Coding sequences of asignal peptide sis present it is not included when calculating percenthomology. In some embodiments, fragment of nucleotide sequence thatencodes fragments of SEQ ID NOs:1-6 or variants thereof may comprisessequences that encode 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 610, 620, 630, 640, 650, 660, 670 ormore contiguous amino acids of any of SEQ ID NOs: 1-6, 67-68 or variantsthereof. In some embodiments, fragment of nucleotide sequence thatencodes fragments of SEQ ID NOs:1-6, 67-68 or variants thereof maysequences that encode comprise 15, 20, 30, 40, 50, 75, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 610, 620, 630, 640, 650, 660,670, 675 or less contiguous amino acids of any of SEQ ID NOs:1-6, 67-68or variants thereof.

In some embodiments, fragments are fragments of SEQ ID NO:64, SEQ IDNO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72.Fragments of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQID NO:70, or SEQ ID NO:72, in each case with or without signal peptidesand/or a methionine at position 1, may comprise 20% or more, 25% ormore, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more,55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% ormore, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more,94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% ormore percent of the length of the particular full length nucleic acidsequence that encodes a filovirus immunogen or variants thereof. Thefragment of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQID NO:70, or SEQ ID NO:72 may comprise a fragment of nucleotide sequencethat encodes polypeptide that is 98% or more homologous, 99% or morehomologous, or 100% identical to the filovirus immunogens encoded by ofSEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, orSEQ ID NO:72 and additionally comprise a signal peptide such as animmunoglobulin signal peptide which is not included when calculatingpercent homology. Fragments of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66,SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72 may comprises sequences thatencode 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 610, 620, 630, 640, 650, 660, 670 or more contiguousamino acids of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69,SEQ ID NO:70, or SEQ ID NO:72 or variants thereof. Fragments of SEQ IDNO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ IDNO:72 may comprises sequences that encode 15, 20, 30, 40, 50, 75, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 610, 620, 630, 640,650, 660, 670, 675 or less contiguous amino acids of SEQ ID NO:64, SEQID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72 orvariants thereof. In some embodiments, fragments are fragments of RNAtranscribed from or encoded by SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66,SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72

m. Identical

“Identical” or “identity” as used herein in the context of two or morenucleic acids or polypeptide sequences, may mean that the sequences havea specified percentage of residues that are the same over a specifiedregion. The percentage may be calculated by optimally aligning the twosequences, comparing the two sequences over the specified region,determining the number of positions at which the identical residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the specified region, and multiplying the result by 100 toyield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of single sequence are included in thedenominator but not the numerator of the calculation. When comparing DNAand RNA, thymine (T) and uracil (U) may be considered equivalent.Identity may be performed manually or by using a computer sequencealgorithm such as BLAST or BLAST 2.0.

n. Impedance

“Impedance” as used herein may be used when discussing the feedbackmechanism and can be converted to a current value according to Ohm'slaw, thus enabling comparisons with the preset current.

o. Immune Response

“Immune response” as used herein may mean the activation of a host'simmune system, e.g., that of a mammal, in response to the introductionof one or more filovirus consensus antigen via the provided DNA plasmidvaccines. The immune response can be in the form of a cellular orhumoral response, or both.

p. Nucleic Acid

“Nucleic acid” or “oligonucleotide” or “polynucleotide” as used hereinmay mean at least two nucleotides covalently linked together. Thedepiction of a single strand also defines the sequence of thecomplementary strand. Thus, a nucleic acid also encompasses thecomplementary strand of a depicted single strand. Many variants of anucleic acid may be used for the same purpose as a given nucleic acid.Thus, a nucleic acid also encompasses substantially identical nucleicacids and complements thereof. A single strand provides a probe that mayhybridize to a target sequence under stringent hybridization conditions.Thus, a nucleic acid also encompasses a probe that hybridizes understringent hybridization conditions.

Nucleic acids may be single stranded or double stranded, or may containportions of both double stranded and single stranded sequence. Thenucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, wherethe nucleic acid may contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosineand isoguanine. Nucleic acids may be obtained by chemical synthesismethods or by recombinant methods.

q. Operably Linked

“Operably linked” as used herein may mean that expression of a gene isunder the control of a promoter with which it is spatially connected. Apromoter may be positioned 5′ (upstream) or 3′ (downstream) of a geneunder its control. The distance between the promoter and a gene may beapproximately the same as the distance between that promoter and thegene it controls in the gene from which the promoter is derived. As isknown in the art, variation in this distance may be accommodated withoutloss of promoter function.

r. Promoter

“Promoter” as used herein may mean a synthetic or naturally-derivedmolecule which is capable of conferring, activating or enhancingexpression of a nucleic acid in a cell. A promoter may comprise one ormore specific transcriptional regulatory sequences to further enhanceexpression and/or to alter the spatial expression and/or temporalexpression of same. A promoter may also comprise distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A promoter may bederived from sources including viral, bacterial, fungal, plants,insects, and animals. A promoter may regulate the expression of a genecomponent constitutively, or differentially with respect to cell, thetissue or organ in which expression occurs or, with respect to thedevelopmental stage at which expression occurs, or in response toexternal stimuli such as physiological stresses, pathogens, metal ions,or inducing agents. Representative examples of promoters include thebacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lacoperator-promoter, tac promoter, SV40 late promoter, SV40 earlypromoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40late promoter and the CMV IE promoter.

s. Stringent Hybridization Conditions

“Stringent hybridization conditions” as used herein may mean conditionsunder which a first nucleic acid sequence (e.g., probe) will hybridizeto a second nucleic acid sequence (e.g., target), such as in a complexmixture of nucleic acids. Stringent conditions are sequence-dependentand will be different in different circumstances. Stringent conditionsmay be selected to be about 5-10° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence at a defined ionic strength pH.The T_(m) may be the temperature (under defined ionic strength, pH, andnucleic concentration) at which 50% of the probes complementary to thetarget hybridize to the target sequence at equilibrium (as the targetsequences are present in excess, at T_(m), 50% of the probes areoccupied at equilibrium). Stringent conditions may be those in which thesalt concentration is less than about 1.0 M sodium ion, such as about0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g.,about 10-50 nucleotides) and at least about 60° C. for long probes(e.g., greater than about 50 nucleotides). Stringent conditions may alsobe achieved with the addition of destabilizing agents such as formamide.For selective or specific hybridization, a positive signal may be atleast 2 to 10 times background hybridization. Exemplary stringenthybridization conditions include the following: 50% formamide, 5×SSC,and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65°C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.

t. Substantially Complementary

“Substantially complementary” as used herein may mean that a firstsequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or99% identical to the complement of a second sequence over a region of 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or morenucleotides or amino acids, or that the two sequences hybridize understringent hybridization conditions.

u. Substantially Identical

“Substantially identical” as used herein may mean that a first andsecond sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98% or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or withrespect to nucleic acids, if the first sequence is substantiallycomplementary to the complement of the second sequence.

v. Variant

“Variant” used herein with respect to a nucleic acid may mean (i) aportion or fragment of a referenced nucleotide sequence; (ii) thecomplement of a referenced nucleotide sequence or portion thereof; (iii)a nucleic acid that is substantially identical to a referenced nucleicacid or the complement thereof; or (iv) a nucleic acid that hybridizesunder stringent conditions to the referenced nucleic acid, complementthereof, or a sequences substantially identical thereto.

“Variant” with respect to a peptide or polypeptide that differs in aminoacid sequence by the insertion, deletion, or conservative substitutionof amino acids, but retain at least one biological activity. Variant mayalso mean a protein with an amino acid sequence that is substantiallyidentical to a referenced protein with an amino acid sequence thatretains at least one biological activity. A conservative substitution ofan amino acid, i.e., replacing an amino acid with a different amino acidof similar properties (e.g., hydrophilicity, degree and distribution ofcharged regions) is recognized in the art as typically involving a minorchange. These minor changes can be identified, in part, by consideringthe hydropathic index of amino acids, as understood in the art. Kyte etal., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an aminoacid is based on a consideration of its hydrophobicity and charge. It isknown in the art that amino acids of similar hydropathic indexes can besubstituted and still retain protein function. In one aspect, aminoacids having hydropathic indexes of ±2 are substituted. Thehydrophilicity of amino acids can also be used to reveal substitutionsthat would result in proteins retaining biological function. Aconsideration of the hydrophilicity of amino acids in the context of apeptide permits calculation of the greatest local average hydrophilicityof that peptide, a useful measure that has been reported to correlatewell with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101,incorporated fully herein by reference. Substitution of amino acidshaving similar hydrophilicity values can result in peptides retainingbiological activity, for example immunogenicity, as is understood in theart. Substitutions may be performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hyrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties. Variants are preferably homologous to SEQ IDNO:1-6,67-68 by 95% or more, 96% or more, 97% or more, 98% or more or99% or more.

“Variant” with respect to a nucleic acid sequence that encodes the samespecific amino acid sequence differs in nucleotide sequence by use ofdifferent codons. Variants of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66,SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72 that encode the same aminoacid sequence as those encoded by SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72 may be any degree ofhomology, preferably 80% or more, 85% or more, 90% or more, 95% or more,96% or more, 97% or more, 98% or more or 99% or more. Variants of RNAtranscribed by SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69,SEQ ID NO:70, or SEQ ID NO:72 that encode the same amino acid sequenceas those encoded by SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ IDNO:69, SEQ ID NO:70, or SEQ ID NO:72 may be any degree of homology,preferably 80% or more, 85% or more, 90% or more, 95% or more, 96% ormore, 97% or more, 98% or more or 99% or more. Variant may also bevariants of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQID NO:70, or SEQ ID NO:72 that encode protein which are variants of theproteins encoded by SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ IDNO:69, SEQ ID NO:70, or SEQ ID NO:72 with an amino acid sequence that issubstantially identical to a referenced protein with an amino acidsequence that retains at least one biological activity, typically theamino acid sequences are homologous by 95% or more, 96% or more, 97% ormore, 98% or more or 99% or more. Variant may also be variants of RNAtranscribed by SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69,SEQ ID NO:70, or SEQ ID NO:72 that encode protein which are variants ofthe proteins encoded the RNA transcribed by SEQ ID NO:64, SEQ ID NO:65,SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72 with an aminoacid sequence that is substantially identical to a referenced proteinwith an amino acid sequence that retains at least one biologicalactivity, typically the amino acid sequences are homologous by 95% ormore, 96% or more, 97% or more, 98% or more or 99% or more

w. Vector

“Vector” used herein may mean a nucleic acid sequence containing anorigin of replication. A vector may be a plasmid, bacteriophage,bacterial, viral vector, artificial chromosome or yeast artificialchromosome. A vector may be a DNA or RNA vector. A vector may be eithera self-replicating extrachromosomal vector or a vector which integratesinto a host genome.

2. Proteins

Provided herein are filovirus immunogens that can be used to inducebroad immunity against multiple subtypes or serotypes of a particularfilovirus antigen. Consensus filovirus antigens may include consensusamino acid sequences of Marburgvirus filovirus glycoprotein MARV RAVimmunogen, consensus amino acid sequences of Marburgvirus filovirusglycoprotein MARV OZO immunogen, consensus amino acid sequences ofMarburgvirus filovirus glycoprotein MARV MUS immunogen, isolate aminoacid sequences of Marburgvirus filovirus glycoprotein MARV ANGimmunogen, consensus amino acid sequences of Zaire ebolavirusglycoprotein ZEBOV immunogen, consensus amino acid sequences of Zaireebolavirus glycoprotein ZEBOV2014 immunogen, isolate amino acidsequences of Zaire ebolavirus glycoprotein ZEBOVCON2 immunogen, andconsensus amino acid sequences of Sudan ebolavirus glycoprotein SUDVimmunogen, respectively. In some embodiments, the immunogens maycomprise a signal peptide from a different protein such as animmunoglobulin protein, for example an IgE signal peptide or an IgGsignal peptide.

The amino acid sequence for immunogens include SEQ ID NO:1-6,67-68variants thereof and fragments of SEQ ID NO:1-6, 67-68 and variantsthereof, optionally including a signal peptide such as for example anIgE or IgG signal peptide.

3. Coding Sequences Encoding Proteins

Coding sequences encoding the proteins set forth herein may be generatedusing routine methods. Composition comprising a nucleic acid sequencethat encodes a consensus Zaire ebolavirus envelope glycoproteinimmunogen, comprising a nucleic acid sequence that encodes a secondconsensus Zaire ebolavirus envelope glycoprotein immunogen, a nucleicacid sequence that encodes a Consensus Zaire ebolavirus Guinea envelopeglycoprotein immunogen envelope glycoprotein immunogen, a nucleic acidsequence that encodes a consensus Sudan ebolavirus envelope glycoproteinimmunogen, a nucleic acid sequence that encodes a Marburg marburgvirusAngola 2005 envelope glycoprotein immunogen are provided and a nucleicacid sequence that encodes a first consensus Marburg marburgvirusenvelope glycoprotein immunogen, a nucleic acid sequence that encodes asecond consensus Marburg marburgvirus envelope glycoprotein immunogen,and a nucleic acid sequence that encodes a third consensus Marburgmarburgvirus envelope glycoprotein immunogen can be generated based uponthe amino acid sequences disclosed.

Nucleic acid sequence may encodes a full length consensus Zaireebolavirus envelope glycoprotein immunogen, a full length secondconsensus Zaire ebolavirus envelope glycoprotein immunogen, a fulllength consensus Consensus Zaire ebolavirus Guinea envelope glycoproteinimmunogen envelope glycoprotein immunogen, a full length consensus Sudanebolavirus envelope glycoprotein immunogen, a full length Marburgmarburgvirus Angola 2005 envelope glycoprotein immunogen, a full lengthfirst consensus Marburg marburgvirus envelope glycoprotein immunogen, afull length second consensus Marburg marburgvirus envelope glycoproteinimmunogen, or a full length third consensus Marburg marburgvirusenvelope glycoprotein immunogen. Nucleic acid sequences may comprise asequence that encodes SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:67, or SEQ ID NO:68. Nucleicacid sequence may comprise SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQID NO:69 SEQ ID NO:70, or SEQ ID NO:72. In one embodiment the nucleotidesequence comprises an RNA sequence transcribed from a DNA sequencedescribed herein. For example, nucleic acids may comprise an RNAsequence transcribed by the DNA sequence of SEQ ID NOs: 64, 65, 66, 69,70 or 72, a fragment thereof or a variant thereof. Nucleic acid sequencemay optionally comprise coding sequences that encode a signal peptidesuch as for example an IgE or IgG signal peptide.

Nucleic acid sequence may encode a fragment of a full length consensusZaire ebolavirus envelope glycoprotein immunogen, a fragment of a fulllength second consensus Zaire ebolavirus envelope glycoproteinimmunogen, a fragment of a full length Consensus Zaire ebolavirus Guineaenvelope glycoprotein immunogen envelope glycoprotein immunogen, afragment of a full length consensus Sudan ebolavirus envelopeglycoprotein immunogen, a fragment of a full length Marburg marburgvirusAngola 2005 envelope glycoprotein immunogen, a fragment of a full lengthfirst consensus Marburg marburgvirus envelope glycoprotein immunogen, afragment of a full length second consensus Marburg marburgvirus envelopeglycoprotein immunogen, or a fragment of a full length third consensusMarburg marburgvirus envelope glycoprotein immunogen. Nucleic acidsequence may comprise a sequence that encodes a fragment of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:67, or SEQ ID NO:68. Nucleic acid sequence may comprise a fragment ofSEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69 SEQ ID NO:70, orSEQ ID NO:72. Fragment sizes are disclosed herein as set forth insection entitled “Fragments”. Nucleic acid sequence may optionallycomprise coding sequences that encode a signal peptide such as forexample an IgE or IgG signal peptide.

Nucleic acid sequences may encode a protein homologous to a full lengthconsensus Zaire ebolavirus envelope glycoprotein immunogen, a fulllength second consensus Zaire ebolavirus envelope glycoproteinimmunogen, a full length Consensus Zaire ebolavirus Guinea envelopeglycoprotein immunogen envelope glycoprotein immunogen, a proteinhomologous to a full length consensus Sudan ebolavirus envelopeglycoprotein immunogen, a protein homologous to a full length Marburgmarburgvirus Angola 2005 envelope glycoprotein immunogen, a proteinhomologous to a full length first consensus Marburg marburgvirusenvelope glycoprotein immunogen, a protein homologous to a full lengthsecond consensus Marburg marburgvirus envelope glycoprotein immunogen,or a protein homologous to a full length third consensus Marburgmarburgvirus envelope glycoprotein immunogen. Nucleic acid sequence maycomprise a sequence that encodes a protein homologous to SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:67, or SEQ ID NO:68. Nucleic acid sequence may be homologous to SEQID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69 SEQ ID NO:70, or SEQID NO:72. Degrees of homology are discussed herein such as in thesection referring to Variants. Nucleic acid sequence may optionallycomprise coding sequences that encode a signal peptide such as forexample an IgE or IgG signal peptide.

Nucleic acid sequence may encode a protein homologous to fragment of afull length consensus Zaire ebolavirus envelope glycoprotein immunogen,a protein homologous to fragment of a full length second consensus Zaireebolavirus envelope glycoprotein immunogen, a protein homologous tofragment of a full length Consensus Zaire ebolavirus Guinea envelopeglycoprotein immunogen envelope glycoprotein immunogen, a proteinhomologous to a fragment of a full length consensus Sudan ebolavirusenvelope glycoprotein immunogen, a protein homologous to a fragment of afull length Marburg marburgvirus Angola 2005 envelope glycoproteinimmunogen, a protein homologous to a fragment of a full length firstconsensus Marburg marburgvirus envelope glycoprotein immunogen, aprotein homologous to a fragment of a full length second consensusMarburg marburgvirus envelope glycoprotein immunogen, or a proteinhomologous to a fragment of a full length third consensus Marburgmarburgvirus envelope glycoprotein immunogen. Nucleic acid sequence maycomprise a sequence that encodes a protein homologous to a fragment ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:67, or SEQ ID NO:68. Nucleic acid sequence may comprisea fragment homologous to SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQID NO:69 SEQ ID NO:70, or SEQ ID NO:72. Degrees of homology arediscussed herein such as in the section referring to Variants. Nucleicacid sequence may optionally comprise coding sequences that encode asignal peptide such as for example an IgE or IgG signal peptide.

SEQ ID NO:64 is the nucleotide sequence insert in plasmid pEBOZ whichencodes consensus Zaire ebolavirus envelope glycoprotein immunogen.

SEQ ID NO:65 is the nucleotide sequence insert in plasmid pEBOS whichencodes consensus Sudan ebolavirus envelope glycoprotein immunogen.

SEQ ID NO:66 is the nucleotide sequence insert in plasmid pMARZ ANGwhich encodes the Marburg marburgvirus Angola 2005 envelopeglycoprotein.

SEQ ID NO:69 is the nucleotide sequence insert in plasmid pEBOZGUI whichencodes consensus Zaire ebolavirus GP envelope glycoprotein immunogen

SEQ ID NO:70 is the nucleotide sequence insert in plasmid pEBOZCON2which encodes a second consensus Zaire ebolavirus envelope glycoprotein

4. Vectors

Vectors include, but are not limited to, plasmids, expression vectors,recombinant viruses, any form of recombinant “naked DNA” vector, and thelike. A “vector” comprises a nucleic acid which can infect, transfect,transiently or permanently transduce a cell. It will be recognized thata vector can be a naked nucleic acid, or a nucleic acid complexed withprotein or lipid. The vector optionally comprises viral or bacterialnucleic acids and/or proteins, and/or membranes (e.g., a cell membrane,a viral lipid envelope, etc.). Vectors include, but are not limited toreplicons (e.g., RNA replicons, bacteriophages) to which fragments ofDNA may be attached and become replicated. Vectors thus include, but arenot limited to RNA, autonomous self-replicating circular or linear DNAor RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No.5,217,879), and include both the expression and non-expression plasmids.Where a recombinant microorganism or cell culture is described ashosting an “expression vector” this includes both extra-chromosomalcircular and linear DNA and DNA that has been incorporated into the hostchromosome(s). Where a vector is being maintained by a host cell, thevector may either be stably replicated by the cells during mitosis as anautonomous structure, or is incorporated within the host's genome.

a. Plasmid

Plasmid may comprise a nucleic acid sequence that encodes one or more ofthe various immunogens disclosed above including coding sequences thatencode synthetic, consensus antigen capable of eliciting an immuneresponse against filoproteins.

A single plasmid may contain coding sequence for a single filoproteinimmunogen, coding sequence for two filoprotein immunogens, codingsequence for three filoprotein immunogens, coding sequence for fourfiloprotein immunogens, coding sequence for five filoprotein immunogensor coding sequence for six filoprotein immunogens. A single plasmid maycontain a coding sequence for a single filoprotein immunogen which canbe formulated together. In some embodiments, a plasmid may comprisecoding sequence that encodes IL-12, IL-15 and/or IL-28.

The plasmid may further comprise an initiation codon, which may beupstream of the coding sequence, and a stop codon, which may bedownstream of the coding sequence. The initiation and termination codonmay be in frame with the coding sequence.

The plasmid may also comprise a promoter that is operably linked to thecoding sequence The promoter operably linked to the coding sequence maybe a promoter from simian virus 40 (SV40), a mouse mammary tumor virus(MMTV) promoter, a human immunodeficiency virus (HIV) promoter such asthe bovine immunodeficiency virus (BIV) long terminal repeat (LTR)promoter, a Moloney virus promoter, an avian leukosis virus (ALV)promoter, a cytomegalovirus (CMV) promoter such as the CMV immediateearly promoter, Epstein Barr virus (EBV) promoter, or a Rous sarcomavirus (RSV) promoter. The promoter may also be a promoter from a humangene such as human actin, human myosin, human hemoglobin, human musclecreatine, or human metalothionein. The promoter may also be a tissuespecific promoter, such as a muscle or skin specific promoter, naturalor synthetic. Examples of such promoters are described in US patentapplication publication no. US20040175727, the contents of which areincorporated herein in its entirety.

The plasmid may also comprise a polyadenylation signal, which may bedownstream of the coding sequence. The polyadenylation signal may be aSV40 polyadenylation signal, LTR polyadenylation signal, bovine growthhormone (bGH) polyadenylation signal, human growth hormone (hGH)polyadenylation signal, or human β-globin polyadenylation signal. TheSV40 polyadenylation signal may be a polyadenylation signal from a pCEP4plasmid (Invitrogen, San Diego, Calif.).

The plasmid may also comprise an enhancer upstream of the codingsequence. The enhancer may be human actin, human myosin, humanhemoglobin, human muscle creatine or a viral enhancer such as one fromCMV, FMDV, RSV or EBV. Polynucleotide function enhances are described inU.S. Pat. Nos. 5,593,972, 5,962,428, and WO94/016737, the contents ofeach are fully incorporated by reference.

The plasmid may also comprise a mammalian origin of replication in orderto maintain the plasmid extrachromosomally and produce multiple copiesof the plasmid in a cell. The plasmid may be pVAX1, pCEP4 or pREP4 fromInvitrogen (San Diego, Calif.), which may comprise the Epstein Barrvirus origin of replication and nuclear antigen EBNA-1 coding region,which may produce high copy episomal replication without integration.The backbone of the plasmid may be pAV0242. The plasmid may be areplication defective adenovirus type 5 (Ad5) plasmid.

The plasmid may also comprise a regulatory sequence, which may be wellsuited for gene expression in a cell into which the plasmid isadministered. The coding sequence may comprise a codon that may allowmore efficient transcription of the coding sequence in the host cell.

The coding sequence may also comprise an Ig leader sequence. The leadersequence may be 5′ of the coding sequence. The consensus antigensencoded by this sequence may comprise an N-terminal Ig leader followedby a consensus antigen protein. The N-terminal Ig leader may be IgE orIgG.

The plasmid may be pSE420 (Invitrogen, San Diego, Calif.), which may beused for protein production in Escherichia coli (E. coli). The plasmidmay also be pYES2 (Invitrogen, San Diego, Calif.), which may be used forprotein production in Saccharomyces cerevisiae strains of yeast. Theplasmid may also be of the MAXBAC™ complete baculovirus expressionsystem (Invitrogen, San Diego, Calif.), which may be used for proteinproduction in insect cells. The plasmid may also be pcDNA I or pcDNA3(Invitrogen, San Diego, Calif.), which may be used for proteinproduction in mammalian cells such as Chinese hamster ovary (CHO) cells.

b. RNA Vectors

In one embodiment, the nucleic acid is an RNA molecule. Accordingly, inone embodiment, the invention provides an RNA molecule encoding one ormore of the envelope glycoprotein (GP) genes of Marburg marburgvirus(MARV), Sudan ebolavirus (SUDV) or Zaire ebolavirus (ZEBOV). The RNA maybe plus-stranded. Accordingly, in some embodiments, the RNA molecule canbe translated by cells without needing any intervening replication stepssuch as reverse transcription. A RNA molecule useful with the inventionmay have a 5′ cap (e.g. a 7-methylguanosine). This cap can enhance invivo translation of the RNA. The 5′ nucleotide of a RNA molecule usefulwith the invention may have a 5′ triphosphate group. In a capped RNAthis may be linked to a 7-methylguanosine via a 5′-to-5′ bridge. A RNAmolecule may have a 3′ poly-A tail. It may also include a poly-Apolymerase recognition sequence (e.g. AAUAAA) near its 3′ end. A RNAmolecule useful with the invention may be single-stranded.

c. Linear Vectors

Also provided herein is a linear nucleic acid vaccine, or linearexpression cassette (“LEC”), that is capable of being efficientlydelivered to a subject via electroporation and expressing one or moredesired antigens. The LEC may be any linear DNA devoid of any phosphatebackbone. The DNA may encode one or more antigens. The LEC may contain apromoter, an intron, a stop codon, a polyadenylation signal. Theexpression of the antigen may be controlled by the promoter. The LEC maynot contain any antibiotic resistance genes and/or a phosphate backbone.The LEC may not contain other nucleic acid sequences unrelated to thedesired antigen gene expression.

The LEC may be derived from any plasmid capable of being linearized. Theplasmid may be capable of expressing the antigen. The plasmid may bepVAX, pcDNA3.0, or provax, or any other expression vector capable ofexpressing the DNA and enabling a cell to translate the sequence to anantigen that is recognized by the immune system.

The LEC may be derived from any plasmid capable of being linearized. Theplasmid may be capable of expressing the antigen. The plasmid may bepVAX, pcDNA3.0, or provax, or any other expression vector capable ofexpressing the DNA and enabling a cell to translate the sequence to aantigen that is recognized by the immune system.

d. Viral Vectors

In one embodiment, viral vectors are provided herein which are capableof delivering a nucleic acid of the invention to a cell. The expressionvector may be provided to a cell in the form of a viral vector. Viralvector technology is well known in the art and is described, forexample, in Sambrook et al. (2001), and in Ausubel et al. (1997), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers. (See, e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

5. Compositions

Compositions are provided which comprise nucleic acid molecules. Thecompositions may comprise a plurality of copies of a single nucleic acidmolecule such a single plasmid, a plurality of copies of two or moredifferent nucleic acid molecules such as two or more different plasmids.For example a composition may comprise plurality of two, three, four,five, six, seven, eight, nine or ten or more different nucleic acidmolecules. Such compositions may comprise plurality of two, three, four,five, six, or more different plasmids.

Compositions may comprise nucleic acid molecules, such as plasmids, thatcollectively contain coding sequence for a single filoprotein immunogenselected from the group consisting of one or more a consensus Zaireebolavirus envelope glycoprotein immunogen, a second consensus Zaireebolavirus envelope glycoprotein immunogen, a Consensus Zaire ebolavirusGuinea envelope glycoprotein immunogen, a consensus Sudan ebolavirusenvelope glycoprotein immunogen, the Marburg marburgvirus Angola 2005envelope glycoprotein, the first consensus Marburg marburgvirus envelopeglycoprotein immunogen, the second consensus Marburg marburgvirusenvelope glycoprotein immunogen and the third consensus Marburgmarburgvirus envelope glycoprotein immunogen.

Composition comprise nucleic acid sequence that encode the combinationof: a consensus Zaire ebolavirus envelope glycoprotein immunogen and aconsensus Sudan ebolavirus envelope glycoprotein immunogen; or aconsensus Zaire ebolavirus envelope glycoprotein immunogen and a secondconsensus Zaire ebolavirus envelope glycoprotein immunogen; or aconsensus Zaire ebolavirus envelope glycoprotein immunogen and aConsensus Zaire ebolavirus Guinea envelope glycoprotein immunogen; or aconsensus Zaire ebolavirus envelope glycoprotein immunogen, a consensusSudan ebolavirus envelope glycoprotein immunogen and the Marburgmarburgvirus Angola 2005 envelope glycoprotein; or a consensus Zaireebolavirus envelope glycoprotein immunogen, a consensus Sudan ebolavirusenvelope glycoprotein immunogen and the second consensus Zaireebolavirus envelope glycoprotein immunogen; or a consensus Zaireebolavirus envelope glycoprotein immunogen, a consensus Sudan ebolavirusenvelope glycoprotein immunogen and the Consensus Zaire ebolavirusGuinea envelope glycoprotein immunogen; or a consensus Zaire ebolavirusenvelope glycoprotein immunogen, a second consensus Zaire ebolavirusenvelope glycoprotein immunogen, the first consensus Marburgmarburgvirus envelope glycoprotein immunogen, the second consensusMarburg marburgvirus envelope glycoprotein immunogen and the thirdconsensus Marburg marburgvirus envelope glycoprotein immunogen; or aconsensus Zaire ebolavirus envelope glycoprotein immunogen, a ConsensusZaire ebolavirus Guinea envelope glycoprotein immunogen, the firstconsensus Marburg marburgvirus envelope glycoprotein immunogen, thesecond consensus Marburg marburgvirus envelope glycoprotein immunogenand the third consensus Marburg marburgvirus envelope glycoproteinimmunogen; or a consensus Zaire ebolavirus envelope glycoproteinimmunogen, a consensus Sudan ebolavirus envelope glycoprotein immunogen,the first consensus Marburg marburgvirus envelope glycoproteinimmunogen, the second consensus Marburg marburgvirus envelopeglycoprotein immunogen and the third consensus Marburg marburgvirusenvelope glycoprotein immunogen; or a consensus Zaire ebolavirusenvelope glycoprotein immunogen, a consensus Sudan ebolavirus envelopeglycoprotein immunogen, the Marburg marburgvirus Angola 2005 envelopeglycoprotein, the first consensus Marburg marburgvirus envelopeglycoprotein immunogen, the second consensus Marburg marburgvirusenvelope glycoprotein immunogen and the third consensus Marburgmarburgvirus envelope glycoprotein immunogen; or a consensus Zaireebolavirus envelope glycoprotein immunogen, a consensus Sudan ebolavirusenvelope glycoprotein immunogen, a Consensus Zaire ebolavirus Guineaenvelope glycoprotein immunogen, the Marburg marburgvirus Angola 2005envelope glycoprotein, the first consensus Marburg marburgvirus envelopeglycoprotein immunogen, the second consensus Marburg marburgvirusenvelope glycoprotein immunogen and the third consensus Marburgmarburgvirus envelope glycoprotein immunogen; or a consensus Zaireebolavirus envelope glycoprotein immunogen, a consensus Sudan ebolavirusenvelope glycoprotein immunogen, a second consensus Zaire ebolavirusenvelope glycoprotein immunogen the Marburg marburgvirus Angola 2005envelope glycoprotein, the first consensus Marburg marburgvirus envelopeglycoprotein immunogen, the second consensus Marburg marburgvirusenvelope glycoprotein immunogen and the third consensus Marburgmarburgvirus envelope glycoprotein immunogen.

Each coding sequence for each filoprotein immunogens is preferablyincluded on a separate plasmid.

Accordingly, compositions that comprise nucleic acid sequence thatencode a consensus Zaire ebolavirus envelope glycoprotein immunogen anda consensus Sudan ebolavirus envelope glycoprotein immunogen; aconsensus Zaire ebolavirus envelope glycoprotein immunogen and a secondconsensus Zaire ebolavirus envelope glycoprotein immunogen; or aconsensus Zaire ebolavirus envelope glycoprotein immunogen and theConsensus Zaire ebolavirus Guinea envelope glycoprotein immunogen, maybe on a single plasmid but are preferably on two separate plasmids.

Compositions that comprise nucleic acid sequence that encode a consensusZaire ebolavirus envelope glycoprotein immunogen, a consensus Sudanebolavirus envelope glycoprotein immunogen and the Marburg marburgvirusAngola 2005 envelope glycoprotein; or a consensus Zaire ebolavirusenvelope glycoprotein immunogen, a consensus Sudan ebolavirus envelopeglycoprotein immunogen and the second consensus Zaire ebolavirusenvelope glycoprotein immunogen; or a consensus Zaire ebolavirusenvelope glycoprotein immunogen, a consensus Sudan ebolavirus envelopeglycoprotein immunogen and the Consensus Zaire ebolavirus Guineaenvelope glycoprotein immunogen; may be on a single plasmid or on twoplasmids in any permutation but are preferably on three separateplasmids.

Compositions that comprise nucleic acid sequence that encode a consensusZaire ebolavirus envelope glycoprotein immunogen, a consensus Sudanebolavirus envelope glycoprotein immunogen, the first consensus Marburgmarburgvirus envelope glycoprotein immunogen, the second consensusMarburg marburgvirus envelope glycoprotein immunogen and the thirdconsensus Marburg marburgvirus envelope glycoprotein immunogen may be ona single plasmid or on two plasmids in any permutation, or on threeplasmids in any permutation or on four plasmids in any permutation butare preferably on five separate plasmids.

Compositions that comprise nucleic acid sequence that encode a consensusZaire ebolavirus envelope glycoprotein immunogen, a second consensusZaire ebolavirus envelope glycoprotein immunogen, the first consensusMarburg marburgvirus envelope glycoprotein immunogen, the secondconsensus Marburg marburgvirus envelope glycoprotein immunogen and thethird consensus Marburg marburgvirus envelope glycoprotein immunogen maybe on a single plasmid or on two plasmids in any permutation, or onthree plasmids in any permutation or on four plasmids in any permutationbut are preferably on five separate plasmids.

Compositions that comprise nucleic acid sequence that encode a consensusZaire ebolavirus envelope glycoprotein immunogen, a Consensus Zaireebolavirus Guinea envelope glycoprotein immunogen, the first consensusMarburg marburgvirus envelope glycoprotein immunogen, the secondconsensus Marburg marburgvirus envelope glycoprotein immunogen and thethird consensus Marburg marburgvirus envelope glycoprotein immunogen maybe on a single plasmid or on two plasmids in any permutation, or onthree plasmids in any permutation or on four plasmids in any permutationbut are preferably on five separate plasmids.

Compositions that comprise nucleic acid sequence that encode a consensusZaire ebolavirus envelope glycoprotein immunogen, a consensus Sudanebolavirus envelope glycoprotein immunogen, a second consensus Zaireebolavirus envelope glycoprotein immunogen the Marburg marburgvirusAngola 2005 envelope glycoprotein, the first consensus Marburgmarburgvirus envelope glycoprotein immunogen, the second consensusMarburg marburgvirus envelope glycoprotein immunogen and the thirdconsensus Marburg marburgvirus envelope glycoprotein immunogen may be ona single plasmid or on two plasmids in any permutation, or on threeplasmids in any permutation or on four plasmids in any permutation or onfour plasmids in any permutation but are preferably on six separateplasmids.

Compositions that comprise nucleic acid sequence that encode a consensusZaire ebolavirus envelope glycoprotein immunogen, a consensus Sudanebolavirus envelope glycoprotein immunogen, the Consensus Zaireebolavirus Guinea envelope glycoprotein immunogen the Marburgmarburgvirus Angola 2005 envelope glycoprotein, the first consensusMarburg marburgvirus envelope glycoprotein immunogen, the secondconsensus Marburg marburgvirus envelope glycoprotein immunogen and thethird consensus Marburg marburgvirus envelope glycoprotein immunogen maybe on a single plasmid or on two plasmids in any permutation, or onthree plasmids in any permutation or on four plasmids in any permutationor on four plasmids in any permutation but are preferably on sixseparate plasmids.

Compositions that comprise nucleic acid sequence that encode a consensusZaire ebolavirus envelope glycoprotein immunogen, a consensus Sudanebolavirus envelope glycoprotein immunogen, the Marburg marburgvirusAngola 2005 envelope glycoprotein, the first consensus Marburgmarburgvirus envelope glycoprotein immunogen, the second consensusMarburg marburgvirus envelope glycoprotein immunogen and the thirdconsensus Marburg marburgvirus envelope glycoprotein immunogen may be ona single plasmid or on two plasmids in any permutation, or on threeplasmids in any permutation or on four plasmids in any permutation or onfive plasmids in any permutation but are preferably on six separateplasmids.

Compositions that comprise nucleic acid sequence that encode a consensusZaire ebolavirus envelope glycoprotein immunogen, a second consensusZaire ebolavirus envelope glycoprotein immunogen, a consensus Sudanebolavirus envelope glycoprotein immunogen, the Marburg marburgvirusAngola 2005 envelope glycoprotein, the first consensus Marburgmarburgvirus envelope glycoprotein immunogen, the second consensusMarburg marburgvirus envelope glycoprotein immunogen and the thirdconsensus Marburg marburgvirus envelope glycoprotein immunogen may be ona single plasmid or on two plasmids in any permutation, or on threeplasmids in any permutation or on four plasmids in any permutation or onfive plasmids in any permutation or on six plasmids in any permutationbut are preferably on seven separate plasmids.

Compositions that comprise nucleic acid sequence that encode a consensusZaire ebolavirus envelope glycoprotein immunogen, the Consensus Zaireebolavirus Guinea envelope glycoprotein immunogen, a consensus Sudanebolavirus envelope glycoprotein immunogen, the Marburg marburgvirusAngola 2005 envelope glycoprotein, the first consensus Marburgmarburgvirus envelope glycoprotein immunogen, the second consensusMarburg marburgvirus envelope glycoprotein immunogen and the thirdconsensus Marburg marburgvirus envelope glycoprotein immunogen may be ona single plasmid or on two plasmids in any permutation, or on threeplasmids in any permutation or on four plasmids in any permutation or onfive plasmids in any permutation or on six plasmids in any permutationbut are preferably on seven separate plasmids.

Likewise, compositions that comprise nucleic acid sequence that encode aconsensus Zaire ebolavirus envelope glycoprotein immunogen, a secondconsensus Zaire ebolavirus envelope glycoprotein immunogen, theConsensus Zaire ebolavirus Guinea envelope glycoprotein immunogen, aconsensus Sudan ebolavirus envelope glycoprotein immunogen, the Marburgmarburgvirus Angola 2005 envelope glycoprotein, the first consensusMarburg marburgvirus envelope glycoprotein immunogen, the secondconsensus Marburg marburgvirus envelope glycoprotein immunogen and thethird consensus Marburg marburgvirus envelope glycoprotein immunogen maybe on a single plasmid or on two plasmids in any permutation, or onthree plasmids in any permutation or on four plasmids in any permutationor on five plasmids in any permutation or on six plasmids in anypermutation or on seven plasmids in any permutation but are preferablyon eight separate plasmids.

6. Vaccine

Provided herein is a vaccine capable of generating in a mammal an immuneresponse against Filovirus, particularly Marburgvirus, Ebolavirus Sudanand/or Ebolavirus Zaire. The vaccine may comprise each plasmid asdiscussed above. The vaccine may comprise a plurality of the plasmids,or combinations thereof. The vaccine may be provided to induce atherapeutic or prophylactic immune response.

Vaccines may be used to deliver nucleic acid molecules that encode aconsensus Zaire ebolavirus envelope glycoprotein immunogen and aconsensus Sudan ebolavirus envelope glycoprotein immunogen. Vaccines maybe used to deliver nucleic acid molecules that encode a consensus Zaireebolavirus envelope glycoprotein immunogen and a Consensus Zaireebolavirus Guinea envelope glycoprotein immunogen. Vaccines may be usedto deliver nucleic acid molecules that encode a consensus Zaireebolavirus envelope glycoprotein immunogen and a second consensus Zaireebola virus envelope glycoprotein immunogen. Vaccines may be used todeliver nucleic acid molecules that encode a consensus Zaire ebolavirusenvelope glycoprotein immunogen, a consensus Sudan ebolavirus envelopeglycoprotein immunogen and a second consensus Zaire ebola virus envelopeglycoprotein immunogen. Vaccines may be used to deliver nucleic acidmolecules that encode a consensus Zaire ebolavirus envelope glycoproteinimmunogen, a consensus Sudan ebolavirus envelope glycoprotein immunogenand the Consensus Zaire ebolavirus Guinea envelope glycoproteinimmunogen. Vaccines may be used to deliver nucleic acid molecules thatencode a consensus Zaire ebolavirus envelope glycoprotein immunogen, aconsensus Sudan ebolavirus envelope glycoprotein immunogen and theMarburg marburgvirus Angola 2005 envelope glycoprotein. Vaccines may beused to deliver nucleic acid molecules that encode a consensus Zaireebolavirus envelope glycoprotein immunogen, a second consensus Zaireebola virus envelope glycoprotein immunogen and the Marburg marburgvirusAngola 2005 envelope glycoprotein. Vaccines may be used to delivernucleic acid molecules that encode a consensus Zaire ebolavirus envelopeglycoprotein immunogen, a Consensus Zaire ebolavirus Guinea envelopeglycoprotein immunogen and the Marburg marburgvirus Angola 2005 envelopeglycoprotein. Vaccines may be used to deliver nucleic acid moleculesthat encode a consensus Zaire ebolavirus envelope glycoproteinimmunogen, a consensus Sudan ebolavirus envelope glycoprotein immunogen,the first consensus Marburg marburgvirus envelope glycoproteinimmunogen, the second consensus Marburg marburgvirus envelopeglycoprotein immunogen and the third consensus Marburg marburgvirusenvelope glycoprotein immunogen. Vaccines may be used to deliver nucleicacid molecules that encode a consensus Zaire ebolavirus envelopeglycoprotein immunogen, a second consensus Zaire ebola virus envelopeglycoprotein immunogen, the first consensus Marburg marburgvirusenvelope glycoprotein immunogen, the second consensus Marburgmarburgvirus envelope glycoprotein immunogen and the third consensusMarburg marburgvirus envelope glycoprotein immunogen. Vaccines may beused to deliver nucleic acid molecules that encode a consensus Zaireebolavirus envelope glycoprotein immunogen, a Consensus Zaire ebolavirusGuinea envelope glycoprotein immunogen, a consensus Sudan ebolavirusenvelope glycoprotein immunogen, the first consensus Marburgmarburgvirus envelope glycoprotein immunogen, the second consensusMarburg marburgvirus envelope glycoprotein immunogen and the thirdconsensus Marburg marburgvirus envelope glycoprotein immunogen. Vaccinesmay be used to deliver nucleic acid molecules that encode a consensusZaire ebolavirus envelope glycoprotein immunogen, a Zaire ebolavirus2014 envelope glycoprotein immunogen, a consensus Sudan ebolavirusenvelope glycoprotein immunogen, the first consensus Marburgmarburgvirus envelope glycoprotein immunogen, the second consensusMarburg marburgvirus envelope glycoprotein immunogen and the thirdconsensus Marburg marburgvirus envelope glycoprotein immunogen. Vaccinesmay be used to deliver nucleic acid molecules that encode a consensusZaire ebolavirus envelope glycoprotein immunogen, a Consensus Zaireebolavirus Guinea envelope glycoprotein immunogen, the first consensusMarburg marburgvirus envelope glycoprotein immunogen, the secondconsensus Marburg marburgvirus envelope glycoprotein immunogen and thethird consensus Marburg marburgvirus envelope glycoprotein immunogen.Vaccines may be used to deliver nucleic acid molecules that encode aconsensus Zaire ebolavirus envelope glycoprotein immunogen, a consensusSudan ebolavirus envelope glycoprotein immunogen, the Marburgmarburgvirus Angola 2005 envelope glycoprotein, the first consensusMarburg marburgvirus envelope glycoprotein immunogen, the secondconsensus Marburg marburgvirus envelope glycoprotein immunogen and thethird consensus Marburg marburgvirus envelope glycoprotein immunogen.Vaccines may be used to deliver nucleic acid molecules that encode aconsensus Zaire ebolavirus envelope glycoprotein immunogen, a secondconsensus Zaire ebola virus envelope glycoprotein immunogen, a consensusSudan ebolavirus envelope glycoprotein immunogen, the Marburgmarburgvirus Angola 2005 envelope glycoprotein, the first consensusMarburg marburgvirus envelope glycoprotein immunogen, the secondconsensus Marburg marburgvirus envelope glycoprotein immunogen and thethird consensus Marburg marburgvirus envelope glycoprotein immunogen.Vaccines may be used to deliver nucleic acid molecules that encode aconsensus Zaire ebolavirus envelope glycoprotein immunogen, theConsensus Zaire ebolavirus Guinea envelope glycoprotein immunogen, aconsensus Sudan ebolavirus envelope glycoprotein immunogen, the Marburgmarburgvirus Angola 2005 envelope glycoprotein, the first consensusMarburg marburgvirus envelope glycoprotein immunogen, the secondconsensus Marburg marburgvirus envelope glycoprotein immunogen and thethird consensus Marburg marburgvirus envelope glycoprotein immunogen.Vaccines may be used to deliver nucleic acid molecules that encode aconsensus Zaire ebolavirus envelope glycoprotein immunogen, a secondconsensus Zaire ebola virus envelope glycoprotein immunogen, theConsensus Zaire ebolavirus Guinea envelope glycoprotein immunogen, aconsensus Sudan ebolavirus envelope glycoprotein immunogen, the Marburgmarburgvirus Angola 2005 envelope glycoprotein, the first consensusMarburg marburgvirus envelope glycoprotein immunogen, the secondconsensus Marburg marburgvirus envelope glycoprotein immunogen and thethird consensus Marburg marburgvirus envelope glycoprotein immunogen.Vaccines are preferably compositions comprising plasmids.

The vaccine may further comprise a pharmaceutically acceptableexcipient. The pharmaceutically acceptable excipient may be functionalmolecules as vehicles, adjuvants, carriers, or diluents. Thepharmaceutically acceptable excipient may be a transfection facilitatingagent, which may include surface active agents, such asimmune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPSanalog including monophosphoryl lipid A, muramyl peptides, quinoneanalogs, vesicles such as squalene and squalene, hyaluronic acid,lipids, liposomes, calcium ions, viral proteins, polyanions,polycations, or nanoparticles, or other known transfection facilitatingagents.

The transfection facilitating agent is a polyanion, polycation,including poly-L-glutamate (LGS), or lipid. The transfectionfacilitating agent is poly-L-glutamate, and more preferably, thepoly-L-glutamate is present in the vaccine at a concentration less than6 mg/ml. The transfection facilitating agent may also include surfaceactive agents such as immune-stimulating complexes (ISCOMS), Freundsincomplete adjuvant, LPS analog including monophosphoryl lipid A,muramyl peptides, quinone analogs and vesicles such as squalene andsqualene, and hyaluronic acid may also be used administered inconjunction with the genetic construct. In some embodiments, the DNAplasmid vaccines may also include a transfection facilitating agent suchas lipids, liposomes, including lecithin liposomes or other liposomesknown in the art, as a DNA-liposome mixture (see for example WO9324640),calcium ions, viral proteins, polyanions, polycations, or nanoparticles,or other known transfection facilitating agents. Preferably, thetransfection facilitating agent is a polyanion, polycation, includingpoly-L-glutamate (LGS), or lipid. Concentration of the transfectionagent in the vaccine is less than 4 mg/ml, less than 2 mg/ml, less than1 mg/ml, less than 0.750 mg/ml, less than 0.500 mg/ml, less than 0.250mg/ml, less than 0.100 mg/ml, less than 0.050 mg/ml, or less than 0.010mg/ml.

The pharmaceutically acceptable excipient may be one or more adjuvants.An adjuvant may be other genes that are expressed from the same or froman alternative plasmid or are delivered as proteins in combination withthe plasmid above in the vaccine. The one or more adjuvants may beproteins and/or nucleic acid molecules that encode proteins selectedfrom the group consisting of: CCL20, α-interferon (IFN-α), β-interferon(IFN-β), γ-interferon, platelet derived growth factor (PDGF), TNFα,TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attractingchemokine (CTACK), epithelial thymus-expressed chemokine (TECK),mucosae-associated epithelial chemokine (MEC), IL-12, IL-15 includingIL-15 having the signal sequence or coding sequence that encodes thesignal sequence deleted and optionally including a different signalpeptide such as that from IgE or coding sequence that encodes adifference signal peptide such as that from IgE, IL-28, MHC, CD80, CD86,IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-1α, MIP-1β, IL-8,L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1,VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF,G-CSF, mutant forms of IL-18, CD40, CD4OL, vascular growth factor,fibroblast growth factor, IL-7, nerve growth factor, vascularendothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1,DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2,DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Re1, MyD88,IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1, JNK, interferon responsegenes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4,RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B,NKG2C, NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof or acombination thereof. In some embodiments adjuvant may be one or moreproteins and/or nucleic acid molecules that encode proteins selectedfrom the group consisting of: CCL-20, IL-12, IL-15, IL-28, CTACK, TECK,MEC or RANTES. Examples of IL-12 constructs and sequences are disclosedin PCT application no. PCT/US1997/019502 and corresponding U.S.application Ser. No. 08/956,865, and U.S. Provisional Application Ser.No. 61/569600 filed Dec. 12, 2011, which are each incorporated herein byreference. Examples of IL-15 constructs and sequences are disclosed inPCT application no. PCT/US04/18962 and corresponding U.S. applicationSer. No. 10/560,650, and in PCT application no. PCT/US07/00886 andcorresponding U.S. application Ser. No. 12/160,766, and in PCTapplication no. PCT/US10/048827, which are each incorporated herein byreference. Examples of IL-28 constructs and sequences are disclosed inPCT application no. PCT/US09/039648 and corresponding U.S. applicationSer. No. 12/936,192, which are each incorporated herein by reference.Examples of RANTES and other constructs and sequences are disclosed inPCT application no. PCT/US1999/004332 and corresponding U.S. applicationSer. No. and 09/622452, which are each incorporated herein by reference.Other examples of RANTES constructs and sequences are disclosed in PCTapplication no. PCT/US11/024098, which is incorporated herein byreference. Examples of RANTES and other constructs and sequences aredisclosed in PCT application no. PCT/US1999/004332 and correspondingU.S. application Ser. No. 09/622452, which are each incorporated hereinby reference. Other examples of RANTES constructs and sequences aredisclosed in PCT application no. PCT/US11/024098, which is incorporatedherein by reference. Examples of chemokines CTACK, TECK and MECconstructs and sequences are disclosed in PCT application no.PCT/US2005/042231 and corresponding U.S. application Ser. No.11/719,646, which are each incorporated herein by reference. Examples ofOX40 and other immunomodulators are disclosed in U.S. application Ser.No. 10/560,653, which is incorporated herein by reference. Examples ofDR5 and other immunomodulators are disclosed in U.S. application Ser.No. 09/622452, which is incorporated herein by reference.

The vaccine may further comprise a genetic vaccine facilitator agent asdescribed in U.S. Ser. No. 021,579 filed Apr. 1, 1994, which is fullyincorporated by reference.

The vaccine may comprise the consensus antigens and plasmids atquantities of from about 1 nanogram to 100 milligrams; about 1 microgramto about 10 milligrams; or preferably about 0.1 microgram to about 10milligrams; or more preferably about 1 milligram to about 2 milligram.In some preferred embodiments, pharmaceutical compositions according tothe present invention comprise about 5 nanogram to about 1000 microgramsof DNA. In some preferred embodiments, the pharmaceutical compositionscontain about 10 nanograms to about 800 micrograms of DNA. In somepreferred embodiments, the pharmaceutical compositions contain about 0.1to about 500 micrograms of DNA. In some preferred embodiments, thepharmaceutical compositions contain about 1 to about 350 micrograms ofDNA. In some preferred embodiments, the pharmaceutical compositionscontain about 25 to about 250 micrograms, from about 100 to about 200microgram, from about 1 nanogram to 100 milligrams; from about 1microgram to about 10 milligrams; from about 0.1 microgram to about 10milligrams; from about 1 milligram to about 2 milligram, from about 5nanogram to about 1000 micrograms, from about 10 nanograms to about 800micrograms, from about 0.1 to about 500 micrograms, from about 1 toabout 350 micrograms, from about 25 to about 250 micrograms, from about100 to about 200 microgram of the consensus antigen or plasmid thereof.

The vaccine may be formulated according to the mode of administration tobe used. An injectable vaccine pharmaceutical composition may besterile, pyrogen free and particulate free. An isotonic formulation orsolution may be used. Additives for isotonicity may include sodiumchloride, dextrose, mannitol, sorbitol, and lactose. The vaccine maycomprise a vasoconstriction agent. The isotonic solutions may includephosphate buffered saline. Vaccine may further comprise stabilizersincluding gelatin and albumin. The stabilizing may allow the formulationto be stable at room or ambient temperature for extended periods of timesuch as LGS or polycations or polyanions to the vaccine formulation.

The vaccine may be stable for is stable at room temperature (25° C.) formore than 1 week, in some embodiments for more than 2 weeks, in someembodiments for more than 3 weeks, in some embodiments for more than 4weeks, in some embodiments for more than 5 weeks, and in someembodiments for more than 6 weeks. In some embodiments, the vaccine isstable for more than one month, more than 2 months, more than 3 months,more than 4 months, more than 5 months, more than 6 months, more than 7months, more than 8 months, more than 9 months, more than 10 months,more than 11 months, or more than 12 months. In some embodiments, thevaccine is stable for more than 1 year, more than 2 years, more thanyears, or more than 5 years. In one embodiment, the vaccine is stableunder refrigeration (2-8° C.). Accordingly, in one embodiment, thevaccine does not require frozen cold-chain. A vaccine is stable if itretains its biological activity for a sufficient period to allow itsintended use (e.g., to generate an immune response in a subject). Forexample, for vaccines that are to be stored, shipped, etc., it may bedesired that the vaccines remain stable for months to years.

7. Methods of Delivery the Vaccine

Provided herein is a method for delivering the vaccine for providinggenetic constructs and proteins of the consensus antigen which compriseepitopes that make them particular effective against immunogens offilovirus, particularly Marburgvirus, Ebolavirus Sudan and/or EbolavirusZaire, against which an immune response can be induced. The method ofdelivering the vaccine or vaccination may be provided to induce atherapeutic and prophylactic immune response. The vaccination processmay generate in the mammal an immune response against filovirus,particularly Marburgvirus, Ebolavirus Sudan and/or Ebolavirus Zaire. Thevaccine may be delivered to an individual to modulate the activity ofthe mammal's immune system and enhance the immune response. The deliveryof the vaccine may be the transfection of the consensus antigen as anucleic acid molecule that is expressed in the cell and delivered to thesurface of the cell upon which the immune system recognized and inducesa cellular, humoral, or cellular and humoral response. The delivery ofthe vaccine may be used to induce or elicit and immune response inmammals against filovirus, particularly Marburgvirus, Ebolavirus Sudanand/or Ebolavirus Zaire by administering to the mammals the vaccine asdiscussed above.

Upon delivery of the vaccine and plasmid into the cells of the mammal,the transfected cells will express and secrete consensus antigens foreach of the plasmids injected from the vaccine. These proteins will berecognized as foreign by the immune system and antibodies will be madeagainst them. These antibodies will be maintained by the immune systemand allow for an effective response to subsequent infections byfilovirus, particularly Marburgvirus, Ebolavirus Sudan and/or EbolavirusZaire.

The vaccine may be administered to a mammal to elicit an immune responsein a mammal. The mammal may be human, primate, non-human primate, cow,cattle, sheep, goat, antelope, bison, water buffalo, bison, bovids,deer, hedgehogs, elephants, llama, alpaca, mice, rats, and chicken.

a. Combination Treatments

The vaccine may be administered in combination with other proteinsand/or genes encoding CCL20, α-interferon, γ-interferon, plateletderived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growthfactor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelialthymus-expressed chemokine (TECK), mucosae-associated epithelialchemokine (MEC), IL-12, IL-15 including IL-15 having the signal sequencedeleted and optionally including the different signal peptide such asthe IgE signal peptide, MHC, CD80, CD86, IL-28, IL-1, IL-2, IL-4, IL-5,IL-6, IL-10, IL-18, MCP-1, MIP-1α, MIP-1β, IL-8, RANTES, L-selectin,P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1,p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, mutantforms of IL-18, CD40, CD4OL, vascular growth factor, fibroblast growthfactor, IL-7, nerve growth factor, vascular endothelial growth factor,Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD,NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun,Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK,SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL,TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40,Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1,TAP2 and functional fragments thereof or combinations thereof. In someembodiments, the vaccine is administered in combination with one or moreof the following nucleic acid molecules and/or proteins: nucleic acidmolecules selected from the group consisting of nucleic acid moleculescomprising coding sequence that encode one or more of CCL20, IL-12,IL-15, IL-28, CTACK, TECK, MEC and RANTES or functional fragmentsthereof, and proteins selected from the group consisting of: CCL02,IL-12 protein, IL-15 protein, IL-28 protein, CTACK protein, TECKprotein, MEC protein or RANTES protein or functional fragments thereof.

The vaccine may be administered by different routes including orally,parenterally, sublingually, transdermally, rectally, transmucosally,topically, via inhalation, via buccal administration, intrapleurally,intravenous, intraarterial, intraperitoneal, subcutaneous,intramuscular, intranasal, intrathecal, and intraarticular orcombinations thereof. For veterinary use, the composition may beadministered as a suitably acceptable formulation in accordance withnormal veterinary practice. The veterinarian can readily determine thedosing regimen and route of administration that is most appropriate fora particular animal. The vaccine may be administered by traditionalsyringes, needleless injection devices, “microprojectile bombardmentgone guns”, or other physical methods such as electroporation (“EP”),“hydrodynamic method”, or ultrasound.

The plasmid of the vaccine may be delivered to the mammal by severalwell-known technologies including DNA injection (also referred to as DNAvaccination) with and without in vivo electroporation, liposomemediated, nanoparticle facilitated, recombinant vectors such asrecombinant adenovirus, recombinant adenovirus associated virus andrecombinant vaccinia. The consensus antigen may be delivered via DNAinjection and along with in vivo electroporation.

b. Electroporation

Administration of the vaccine via electroporation of the plasmids of thevaccine may be accomplished using electroporation devices that can beconfigured to deliver to a desired tissue of a mammal a pulse of energyeffective to cause reversible pores to form in cell membranes, andpreferable the pulse of energy is a constant current similar to a presetcurrent input by a user. The electroporation device may comprise anelectroporation component and an electrode assembly or handle assembly.The electroporation component may include and incorporate one or more ofthe various elements of the electroporation devices, including:controller, current waveform generator, impedance tester, waveformlogger, input element, status reporting element, communication port,memory component, power source, and power switch. The electroporationmay be accomplished using an in vivo electroporation device, for exampleCELLECTRA EP system (VGX Pharmaceuticals, Blue Bell, Pa.) or Elgenelectroporator (Genetronics, San Diego, Calif.) to facilitatetransfection of cells by the plasmid.

The electroporation component may function as one element of theelectroporation devices, and the other elements are separate elements(or components) in communication with the electroporation component. Theelectroporation component may function as more than one element of theelectroporation devices, which may be in communication with still otherelements of the electroporation devices separate from theelectroporation component. The elements of the electroporation devicesexisting as parts of one electromechanical or mechanical device may notlimited as the elements can function as one device or as separateelements in communication with one another. The electroporationcomponent may be capable of delivering the pulse of energy that producesthe constant current in the desired tissue, and includes a feedbackmechanism. The electrode assembly may include an electrode array havinga plurality of electrodes in a spatial arrangement, wherein theelectrode assembly receives the pulse of energy from the electroporationcomponent and delivers same to the desired tissue through theelectrodes. At least one of the plurality of electrodes is neutralduring delivery of the pulse of energy and measures impedance in thedesired tissue and communicates the impedance to the electroporationcomponent. The feedback mechanism may receive the measured impedance andcan adjust the pulse of energy delivered by the electroporationcomponent to maintain the constant current.

A plurality of electrodes may deliver the pulse of energy in adecentralized pattern. The plurality of electrodes may deliver the pulseof energy in the decentralized pattern through the control of theelectrodes under a programmed sequence, and the programmed sequence isinput by a user to the electroporation component. The programmedsequence may comprise a plurality of pulses delivered in sequence,wherein each pulse of the plurality of pulses is delivered by at leasttwo active electrodes with one neutral electrode that measuresimpedance, and wherein a subsequent pulse of the plurality of pulses isdelivered by a different one of at least two active electrodes with oneneutral electrode that measures impedance.

The feedback mechanism may be performed by either hardware or software.The feedback mechanism may be performed by an analog closed-loopcircuit. The feedback occurs every 50 μs, 20 μs, 10 μs or 1 μs, but ispreferably a real-time feedback or instantaneous (i.e., substantiallyinstantaneous as determined by available techniques for determiningresponse time). The neutral electrode may measure the impedance in thedesired tissue and communicates the impedance to the feedback mechanism,and the feedback mechanism responds to the impedance and adjusts thepulse of energy to maintain the constant current at a value similar tothe preset current. The feedback mechanism may maintain the constantcurrent continuously and instantaneously during the delivery of thepulse of energy.

Examples of electroporation devices and electroporation methods that mayfacilitate delivery of the DNA vaccines of the present invention,include those described in U.S. Pat. No. 7,245,963 by Draghia-Akli, etal., U.S. Patent Pub. 2005/0052630 submitted by Smith, et al., thecontents of which are hereby incorporated by reference in theirentirety. Other electroporation devices and electroporation methods thatmay be used for facilitating delivery of the DNA vaccines include thoseprovided in co-pending and co-owned U.S. Patent application, Ser. No.11/874072, filed Oct. 17, 2007, which claims the benefit under 35 USC119(e) to U.S. Provisional Applications Ser. Nos. 60/852,149, filed Oct.17, 2006, and 60/978,982, filed Oct. 10, 2007, all of which are herebyincorporated in their entirety.

U.S. Pat. No. 7,245,963 by Draghia-Akli, et al. describes modularelectrode systems and their use for facilitating the introduction of abiomolecule into cells of a selected tissue in a body or plant. Themodular electrode systems may comprise a plurality of needle electrodes;a hypodermic needle; an electrical connector that provides a conductivelink from a programmable constant-current pulse controller to theplurality of needle electrodes; and a power source. An operator cangrasp the plurality of needle electrodes that are mounted on a supportstructure and firmly insert them into the selected tissue in a body orplant. The biomolecules are then delivered via the hypodermic needleinto the selected tissue. The programmable constant-current pulsecontroller is activated and constant-current electrical pulse is appliedto the plurality of needle electrodes. The applied constant-currentelectrical pulse facilitates the introduction of the biomolecule intothe cell between the plurality of electrodes. The entire content of U.S.Pat. No. 7,245,963 is hereby incorporated by reference.

U.S. Patent Pub. 2005/0052630 submitted by Smith, et al. describes anelectroporation device which may be used to effectively facilitate theintroduction of a biomolecule into cells of a selected tissue in a bodyor plant. The electroporation device comprises an electro-kinetic device(“EKD device”) whose operation is specified by software or firmware. TheEKD device produces a series of programmable constant-current pulsepatterns between electrodes in an array based on user control and inputof the pulse parameters, and allows the storage and acquisition ofcurrent waveform data. The electroporation device also comprises areplaceable electrode disk having an array of needle electrodes, acentral injection channel for an injection needle, and a removable guidedisk. The entire content of U.S. Patent Pub. 2005/0052630 is herebyincorporated by reference.

The electrode arrays and methods described in U.S. Pat. No. 7,245,963and U.S. Patent Pub. 2005/0052630 may be adapted for deep penetrationinto not only tissues such as muscle, but also other tissues or organs.Because of the configuration of the electrode array, the injectionneedle (to deliver the biomolecule of choice) is also insertedcompletely into the target organ, and the injection is administeredperpendicular to the target issue, in the area that is pre-delineated bythe electrodes The electrodes described in U.S. Pat. No. 7,245,963 andU.S. Patent Pub. 2005/005263 are preferably 20 mm long and 21 gauge.

Additionally, contemplated in some embodiments that incorporateelectroporation devices and uses thereof, there are electroporationdevices that are those described in the following patents: U.S. Pat. No.5,273,525 issued Dec. 28, 1993, U.S. Pat. No. 6,110,161 issued Aug. 29,2000, U.S. Pat. No. 6,261,281 issued Jul. 17, 2001, and U.S. Pat. No.6,958,060 issued Oct. 25, 2005, and U.S. Pat. No. 6,939,862 issued Sep.6, 2005. Furthermore, patents covering subject matter provided in U.S.Pat. No. 6,697,669 issued Feb. 24, 2004, which concerns delivery of DNAusing any of a variety of devices, and U.S. Pat. No. 7,328,064 issuedFeb. 5, 2008, drawn to method of injecting DNA are contemplated herein.The above-patents are incorporated by reference in their entirety.

c. Method of Preparing DNA Plasmids

Provided herein is methods for preparing the DNA plasmids that comprisethe DNA vaccines discussed herein. The DNA plasmids, after the finalsubcloning step into the mammalian expression plasmid, can be used toinoculate a cell culture in a large scale fermentation tank, using knownmethods in the art.

The DNA plasmids for use with the EP devices of the present inventioncan be formulated or manufactured using a combination of known devicesand techniques, but preferably they are manufactured using an optimizedplasmid manufacturing technique that is described in a licensed,co-pending U.S. provisional application U.S. Ser. No. 60/939,792, whichwas filed on May 23, 2007. In some examples, the DNA plasmids used inthese studies can be formulated at concentrations greater than or equalto 10 mg/mL. The manufacturing techniques also include or incorporatevarious devices and protocols that are commonly known to those ofordinary skill in the art, in addition to those described in U.S. Ser.No. 60/939792, including those described in a licensed patent, U.S. Pat.No. 7,238,522, which issued on Jul. 3, 2007. The above-referencedapplication and patent, U.S. Ser. No. 60/939,792 and U.S. Pat. No.7,238,522, respectively, are hereby incorporated in their entirety.

EXAMPLES

The present invention is further illustrated in the following Example.It should be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, various modifications of the invention in addition tothose shown and described herein will be apparent to those skilled inthe art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Example 1 Methods Plasmid Vaccine Construction

The pMARV, pEBOS, and pEBOZ plasmid DNA constructs encode full-length GPproteins. An amino acid consensus strategy was used for the pEBOS andpEBOZ, while a type-matched sequence from the 2005 Angola outbreakstrain was used (GenBank #VGP_MABVR) for pMARV (Towner J S, et al.(2006). Marburgvirus genomics and association with a large hemorrhagicfever outbreak in Angola. J Virol 80: 6497-6516). Consensus sequenceswere determined by alignment of the prevailing ZEBOV and SUDV GP aminoacid sequences and generating a consensus for each. Each vaccine GP genewas genetically optimized for expression in humans (including codon- andRNA-optimization for enhancing protein expression (GenScript,Piscataway, N.J.)), synthesized commercially, and then subcloned(GenScript, Piscataway, N.J.) into modified pVAX1 mammalian expressionvectors (Invitrogen, Carlsbad, Calif.) under the control of thecytomegalovirus immediate-early (CMV) promoter; modifications include2A>C, 3C>T, 4T>G, 241C>G, 1,942C>T, 2,876A>-, 3,277C>T, and 3,753G>C.Phylogenetic analysis was performed by multiple-alignment with ClustalWusing MEGA version 5 software. Alternatively, GP diversity among theMARV was much higher (˜70% identity) in comparison, so a consensusstrategy was not adopted. For coverage of MARV, we chose to utilize theMGP sequence from the 2005 outbreak in Angola (GenBank #VGP_MABVR) sinceit was solely responsible for the largest and deadliest MARV outbreak todate. This sequence was greater than 10% divergent from either of itsclosest cluster of relative strains including Musoke, Popp and Leiden(10.6% divergence), or Uganda (01Uga07), Durba (05DRC99 and 07DRC99) andOzolin (10.3% divergence). Altogether, a three-plasmid strategy formedthe foundation for our novel trivalent polyvalent-filovirus vaccinestrategy.

Transfections and Immunoblotting

Human Embryonic Kidney (HEK) 293T cells were cultured, transfected, andharvested. Briefly, cells were grown in DMEM with 10% FBS, 1% Pen-strep,sodium pyruvate, and L-glutamine. Cells were cultured in 150 mm Corningdishes and grown to 70% confluence overnight in a 37° incubator with 5%CO₂. Dishes were transfected with 10-25 μg of Filoviridae pDNA usingeither a Calphos™ Mammalian Transfection Kit protocol (Clonetech) orLipofectamine™ 2000 reagent (Invitrogen) per the manufacturer's protocoland then incubated for 24-48 h. Cells were harvested with ice cold PBS,centrifuged and washed, and then pelleted for Western immunoblot or FACSanalysis. Standard Western blotting was used and GP-specific MAbs forGP1 detection were generated. Data from Western immunoblottingexperiments is shown in FIG. 1B. Data from FACS analysis is shown inFIG. 1C.

Animals, Vaccinations, and Challenge

Adult female C57BL/6 (H-2^(b)), BALB/cJ (H-2^(d)), and B10.Br (H-2^(k))mice were purchased from The Jackson Laboratory (Bar Harbor, Me.) whileHartley guinea pigs were from Charles River (Wilmington, Mass.). Allanimal experimentation was conducted following UPenn IACUC and School ofMedicine Animal Facility, or NML Institutional Animal Care Committee ofthe PHAC and the Canadian Council on Animal Care guidelines for housingand care of laboratory animals and performed in accordance withrecommendations in the Guide for the Care and Use of Laboratory Animalsof NIH after pertinent review and approval by the abovementionedinstitutions. UPenn and NML comply with NIH policy on animal welfare,the Animal Welfare Act, and all other applicable federal, state andlocal laws.

Mice were immunized i.m. by needle injection with 40 μg of plasmidresuspended in water, while guinea pigs were immunized i.d., with 200 μgof each into three separate vaccination sites. Vaccinations wereimmediately followed by EP at the same site. Briefly, a three-prongedCELLECTRA® adaptive constant current Minimally Invasive Device wasinserted approximately 2 mm i.d. (Inovio Pharmaceuticals, Inc., BlueBell, Pa.). Square-wave pulses were delivered through a triangular3-electrode array consisting of 26-gauge solid stainless steelelectrodes and two constant-current pulses of 0.1 Amps were deliveredfor 52 msec/pulse separated by a 1 sec delay.

For lethal challenge studies, challenges were limited to rodent-adaptedZEBOV and MARV. Guinea pigs were challenged 28 days after the finalvaccination by i.p. injection with 1,000 LD₅₀ of guinea pig-adaptedZEBOV (21.3 FFU/animal) (Richardson J S, Abou M C, Tran K N, Kumar A,Sahai B M, Kobinger G P (2011). Impact of systemic or mucosal immunityto adenovirus on ad-based Ebola virus vaccine efficacy in guinea pigs. JInfect Dis 204 Suppl 3: S1032-1042) or 1,000 LD₅₀ MARV-Angola (681TCID50/animal). Briefly, the guinea-pig adapted MARV was made by theserial passage of wild-type MARV-Angola in outbred adult female Hartleyguinea pigs. Seven days after inoculation, the animals were euthanizedand livers were harvested and homogenized. This homogenate was theninjected i.p. into naive adult guinea pigs and the process repeateduntil animals lost weight, gloss of hair, and succumbed to infectionsimilar to EBOV adaptation in guinea pigs. For mouse lethal challengestudies (Kobinger G P, et al. (2006). Chimpanzee adenovirus vaccineprotects against Zaire Ebola virus. Virology 346: 394-401), mice wereinjected i.p. with 200 μl of a 1,000 LD₅₀ (10 FFU/animal) ofmouse-adapted ZEBOV. All animals were weighed daily and monitored fordisease progression using an approved score sheet for at least 18 daysfor mice and 22 days for guinea pigs. All infectious work was performedin a ‘Biosafety Level 4’ (BSL4) facility at NML, PHAC.

ELISA and Neutralization Assays

Antibody (Ab) titers were determined using 96-well ELISA plates coatedwith either sucrose-purified MARV Ozolin GP or ZGP, or with negativecontrol sucrose-purified Nipah G protein at a concentration of 1:2,000.Briefly, the plates were then incubated for 18 hat 4° C., washed withPBS and 0.1% Tween-20, and 100 μl/sample of the sera were tested intriplicate (at dilutions 1:100, 1:400, 1:1,600, and 1:6,400 in PBS with5% skim milk and 0.5% Tween-20). Following an incubation at 37° C. for 1h in a moist container, the plates were washed and then 100 μl of goatanti-mouse IgG-conjugated HRP antibody (Cedarlane) was added (1:2,000dilution) and incubated for another 37° C. for 1 h in a moist container.After a wash, 100 μl of the ABST(2,2′-azino-bis(3-ethylbenthiazoline-6-sulphonic acid) and peroxidasesubstrate (Cedarlane) was added to visualize Ab binding. Again in amoist container, the plate was incubated for 30 min at 37° C. and thenlater read at 405 nm. Positive binding results were characterized bybeing >3 SD when subtracting the positive control from the negativecontrol serum.

The ZEBOV neutralization assay was performed. Briefly, Sera collectedfrom immunized mice and guinea pigs were inactivated at 56° C. for 45minutes and serial dilutions of each sample (1:20,1:40, etc. . . . , formice and 1:50 for guinea pigs, in 50 μl of DMEM) was mixed with equalvolume of ZEBOV expressing the EGFP reporter gene (ZEBOV-EGFP) (100transducing units/well, according to EGFP expression) and incubated at37° C. for 90 minutes. The mixture was then transferred ontosub-confluent VeroE6 cells in 96-well flat-bottomed plates and incubatedfor 5-10 minutes at RT. Control wells were infected with equal amountsof the ZEBOV-EGFP virus without addition of serum or with non-immuneserum. 100 μl of DMEM supplemented with 20% FBS was then added to eachwell and plates were incubated at 37° C. in 5% CO₂ for 48 h.

Alternatively, neutralization of MARV-Angola 368 was assessed using animmunofluorescent assay. A primary rabbit anti-MARV Ab and secondarygoat anti-rabbit IgG FITC-conjugated Ab was used for detection.Neutralizing Abs (NAbs) against SUDV Boniface were assayed based oncytopathic effect (CPE) on CV-1 cells. Cells were incubated with equalparts of immunized sera and SUDV Boniface for 10 days beforesubsequently fixed with 10% buffered formalin for 24 hours and examinedunder a light microscope. EGFP and FITC positive cells were counted ineach well and sample dilutions showing >50% reduction in the number ofgreen cells compared to controls scored positive for NAb. Alternatively,NAbs against SUDV-Boniface were assayed based on cytopathic effect (CPE)on CV-1 cells. All infectious work was performed in the BSL4 laboratoryof NML, PHAC.

Splenocyte isolation

Mice were sacrificed 8-11 days following the final immunization and thespleens were harvested. Briefly, spleens were placed in RPMI 1640 medium(Mediatech Inc., Manassas, Va.) supplemented with 10% FBS, 1× Anti-anti(Invitrogen), and 1×3-ME (Invitrogen). Splenocytes were isolated bymechanical disruption of the spleen using a Stomacher machine (SewardLaboratory Systems Inc., Bohemia, N.Y.), and the resulting product wasfiltered using a 40 μm cell strainer (BD Falcon). The cells were thentreated for 5 min with ACK lysis buffer (Lonza, Switzerland) for lysisof RBCs, washed in PBS, and then resuspended in RPMI medium for use inELISPOT or FACS assay.

ELISPOT Assays

Standard IFNγ ELISPOT assay was performed. Briefly, 96-well plates(Millipore, Billerica, Mass.) were coated with anti-mouse IFN-γ captureantibody and incubated for 24 h at 4° C. (R&D Systems, Minneapolis,Minn.). The following day, plates were washed with PBS and then blockedfor 2 h with blocking buffer (1% BSA and 5% sucrose in PBS). Splenocytes(1-2×10⁵ cells/well) were plated in triplicate and stimulated overnightat 37° C. in 5% CO₂ and in the presence of either RPMI 1640 (negativecontrol), Con A (positive control), or GP peptides either individually(15-mers overlapping by 9 amino acids and spanning the lengths of theirrespective GP) or whole pooled (2.5 μg/ml final). After 18-24 h ofstimulation, the plates were washed in PBS and then incubated for 24 hat 4° C. with biotinylated anti-mouse IFN-γ mAb (R&D Systems,Minneapolis, Minn.). Next, the plates were washed again in PBS, andstreptavidin-alkaline phosphatase (MabTech, Sweden) was added to eachwell and incubated for 2 h at RT. Lastly, the plates were washed againin PBS and then BCIP/NBT Plus substrate (MabTech) was added to each wellfor 5-30 min for spot development. As soon as the development processwas complete upon visual inspection, the plate were rinsed withdistilled water and then dried overnight at RT. Spots were enumeratedusing an automated ELISPOT reader (Cellular Technology Ltd., ShakerHeights, Ohio).

For comprehensive analysis of T cell breadth, standard IFNγ ELISPOT wasmodified herein as previously described in Shedlock D J, et al. (2012).SUPRA. Identification and measurement of subdominant and immunodominantT cell epitopes were assessed by stimulating splenocytes with individualpeptides as opposed to whole or matrix peptide pools; the traditionalpractice of pooling peptides for the sake of sample preservation, suchas the use of matrix array pools, results in a reduction of assaysensitivity since total functional responses in pools containingmultiple epitope-displaying peptides will effectively lower assayresolution, i.e. ‘drown-out’ those of lower magnitude. Thus, modifiedELISPOT was performed with individual peptides (15-mers overlapping by 9amino acids; 2.5 μg/ml final) spanning each GP immunogen. Peptidescontaining T cell epitopes were identified (≥10 AVE IFNγ+ spots AND ≥80%animal response rate; summarized in Tables 1-6) and then later confirmedfunctionally and phenotypically by FACS. No shared or partial epitopeswere identified, nor did FACS data or web-based epitope predictionsoftware suggest the presence of a CD4+ or CD8+ T cell epitope that waspreserved within consecutive peptides. Here, possible shared/partial Tcell epitopes were addressed for all instances of contiguous peptideresponses as identified by modified ELISPOT assay. Cells were stimulatedindividually with each of the contiguous peptides, as well as paired incombination for direct comparison, and were defined as ‘shared/partial’if the combined response was not greater than either of the twoindividual responses. Also, it must be noted, that the epitopic responsepresented herein may not have been completely comprehensive since the‘15-mer overlapping by 9 amino acids’ algorithm for generating peptidesis biased towards complete coverage of CD8 T cell epitopes which mayunderestimate CD4 T cell responses due to the nature of classII-restricted epitopes being longer than 15 amino acids. Lastly, aminoacid similarity plots were generated using Vector NTI software and theresults are shown in FIG. 4B.

Flow Cytometry

Splenocytes were added to a 96-well plate (1×10⁶ cells/well) andstimulated for 5-6 h with either individual peptides or ‘Minimal PeptidePools’ (2.5 μg/ml final). Individual peptides stimulation was used forfunctional confirmation of all peptides identified by modified ELISPOT(Tables 1-6) as well as phenotypic characterization. Splenocytes andtransfected 293Ts were first pre-stained with LIVE/DEAD® Fixable VioletDead Cell Stain Kit (Invitrogen). For splenocytes, cells weresurface-stained for CD19 (V450; clone 1D3), CD4 (PE-Cy7; clone RM4-5),CD8α (APC-Cy7; clone 53-6.7) and CD44 (PE-Cy5; clone IM7) (BDBiosciences, San Jose, Calif.), washed three times in PBS+1% FBS,permeabilized with BD Cytofix/Cytoperm™ kit, and then stainedintracellularly with IFNγ (APC; clone XMG1.2), TNF (FITC; cloneMP6-XT22), CD3 (PE-cy5.5; clone 145-2C11), and T-bet (PE; clone 4B10)(eBioscience, San Diego, Calif.). GP expression in transfected 293Tcells was assessed 24 h post-transfection. Indirect staining wasperformed following a 30 min incubation at 4° C. in PBS+1% FBScontaining the indicated mouse-derived GP-specific polyclonal serumreagent (1:200 dilution), each produced by pooling serum from H-2^(b)mice immunized three times with their respective DNA vaccine or pVAX1empty vector control. Cells were then stained with FITC-conjugated goatanti-mouse IgG (BioLegend, San Diego, Calif.), washed extensively, andthen stained for MHC class I (HLA-ABC; PE-Cy7; clone G46-2.6; BD). Allcells were fixed in 1% paraformaldehyde. All data was collected using aLSRII flow cytometer (BD) and analyzed using FlowJo software (Tree Star,Ashland, Oreg.). Splenocytes were gated for activated IFNγ-producing Tcells that were CD3+ CD44+, CD4+ or CD8+, and negative for the B cellmarker CD19 and viability dye.

FIG. 6 shows a GP-specific T cell gating. Functional and phenotypicanalysis for peptides containing T cell epitopes as identified byELISPOT was performed by FACS gating of total lymphocytes, live (LD)CD3+ cells that were negative for CD 19 and LIVE-DEAD (dump channel),singlets (excludes cell doublets), CD4+ and CD8+ cells, activated cells(CD44+), and peptide-specific IFNγ-producing T cells.

Statistical Analysis

Significance for unrooted phylogenetic trees was determined bymaximum-likelihood method and verified by bootstrap analysis andsignificant support values (≥80%; 1,000 bootstrap replicates) weredetermined by MEGA version 5 software. Group analyses were completed bymatched, two-tailed, unpaired t test and survival curves were analyzedby log-rank (Mantel-Cox) test. All values are mean±SEM and statisticalanalyses were performed by GraphPad Prism (La Jolla, Calif.).

Results Vaccine Construction and Expression

Phylogenetic analysis revealed relative conservation among the EBOV GPs(94.4% for SUDV and 92.9% for ZEBOV), whereas the MARV GP (MGP) weremore divergent (-70% conserved). Thus, a consensus strategy, asdetermined by alignment of the prevailing ZEBOV and SUDV GP amino acidsequences, was adopted for the EBOV GPs, while a type-matched strategywas used for MARV employing the 2005 Angola outbreak sequence which wassolely responsible for the largest and deadliest MARV outbreak. Each GPtransgene was genetically optimized, synthesized commercially, and thensubcloned into modified pVAX1 mammalian expression vector. Altogether, athree-plasmid strategy formed the foundation for our novelpolyvalent-filovirus vaccine strategy.

HEK 293T cells were transfected separately with each plasmid and GPexpression was assessed by Western immunoblotting and FACS. A ˜130 kDaprotein was observed for each in cell lysates harvested 48 hpost-transfection using species-specific anti-GP1 mAbs for detectionResults are shown in FIG. 1B. For a comparative control, recombinantvesicular stomatitis viruses (rVSV) expressing the respective GPs wereloaded in concurrent lanes. Next, GP expression on the cell surface wasanalyzed 24 h post-transfection by indirect staining with GP-specific orcontrol polyclonal serum by FACS. Results are shown in FIG. 1C. Cellsurface expression was detected for all vaccine plasmids while littlenon-specific binding was observed; control serum did not react withGP-transfected cells nor did the positive sera with pVAX1-transfectedcells (data shown for pEBOZ). As expected for the EBOV GPs, cell surfaceexpression sterically occluded recognition of surface MHC class I, aswell as β1-integrin (Francica J R, Varela-Rohena A, Medvec A, Plesa G,Riley J L, Bates P (2010). Steric shielding of surface epitopes andimpaired immune recognition induced by the ebola virus glycoprotein.PLoS Pathog 6: e1001098).

Complete Protection Against MARV and ZEBOV Challenge

To determine protective efficacy, we employed the guinea pig preclinicalchallenge model. Preclinical immunogenicity and efficacy studies wereperformed herein using the guinea pig and mouse models. The guinea pigpreclinical model has been extensively used as a screening and‘proof-of-concept’ tool for filoviral vaccine development. Althoughprimary isolates of MARV and EBOV cause non-fatal illness in guineapigs, a small number of passages in this host results in selection ofvariants able to cause fatal disease with pathological features similarto those seen in filovirus-infected primates. Similarly, mice have alsobeen widely used for filoviral vaccine development, however, unlike theguinea pig model, immunodetection reagents for assessing immunity and Tcell responses are extensively available. Infection with amurine-adapted ZEBOV (mZEBOV) results in disease characterized by highlevels of virus in target organs and pathologic changes in livers andspleens akin to those found in EBOV-infected primates.

Guinea pigs (n=24) were immunized i.d. two times with 200 μg of eachplasmid (pEBOZ, pEBOS and pMARV) into three separate vaccination sitesor with pVAX1 empty vector control (n=9), and then boosted with the samevaccines one month later. Animals were challenged 28 days following thesecond immunization with 1,000 LD₅₀ of a guinea pig-adapted MARV-Angola(gpMARV) (n=9) or ZEBOV (gpZEBOV) (n=15) in a BSL-4 facility, and thenobserved and weighed daily. Results are shown in FIGS. 2A-2H. Vaccinatedanimals were completely protected while control-vaccinated animalssuccumbed to gpMARV by 10 days post-challenge (n=3; P=0.0052) or togpZEBOV by day 7 post-challenge (n=6; P=0.0008) (FIG. 2A and FIG. 2E).Additionally, vaccinated animals were protected from weight loss (FIG.2B and FIG. 2F; P<0.0001). It is likely that vaccine-induced Abs mayhave contributed to protection since GP-specific Abs in pooled serumexhibited a significant increase in binding (FIG. 2C and FIG. 2G) andneutralization (FIG. 2D and FIG. 2H) titers. Experiments were performedin a BSL-4 facility and repeated twice with similar results and errorbars in Figures A-2H represent SEM. Group analyses were completed bymatched, two-tailed, unpaired t test and survival curves were analyzedby log-rank (Mantel-Cox) test.

Plasmid Vaccines were Highly Immunogenic

To better characterize immune correlates as driven by the protective DNAvaccine (plasmids pEBOZ, pEBOS and pMARV, also referred to as trivalentDNA vaccine), we next employed the mouse model which has been widelyused as a screening and ‘proof-of-concept’ tool for filoviral vaccinedevelopment and in which extensive immunodetection reagents areavailable. First, B cell responses were assessed in H-2^(d) mice(n=5/group) 20 days following each of two vaccinations, three weeksbetween injections with 40 μg of respective monovalent DNA vaccine. Datafrom these experiments is shown in FIGS. 3A-3C. While little GP-specificIgG was observed in pre-bleed control samples, as shown in FIG. 3A andFIG. 3B, a significant increase was detected in all animals followingvaccination. Since purified SGP was not available, purified ZGP was usedas a surrogate. IgG in SUDV-vaccinated mice bound ZGP, demonstrating theability for vaccine-induced Ab generation as well as its capability forcross-species recognition. Additionally, seroconversion occurred in 100%of vaccinated animals after only one immunization, after which responseswere significantly increased by homologous boost; AVE reciprocalendpoint dilution titres were boosted 22.1-fold in pMARV-immunized mice,and 3.4-fold and 8.6-fold in pEBOS- and pEBOZ-vaccinated animals,respectively. Samples were next assayed for neutralization of ZEBOV,SUDV-Boniface, and MARV-Angola in a BSL-4 facility. The results of theneutralization assay are shown in FIG. 3C. Significant increases in NAbtitres were detected following vaccination in all animals.

Mice from two different genetic backgrounds (H-2^(d) and H-2^(b);n=5/group) were immunized with 40 μg of respective plasmids pEBOZ, pEBOSand pMARV, homologous boosted after two weeks, and then sacrificed 8days later for T cell analysis Results from a novel modified ELISPOTassay to assess the comprehensive vaccine-induced T cell response, inwhich splenocytes were stimulated using individual peptides as opposedto matrix pools are shown in FIG. 4A. DNA vaccination induced robustIFNγ+ responses that recognized a diversity of T-cell epitopes (Tables1-6). All positive epitope-comprising peptides were subsequently gated(See FIG. 6), confirmed, and further characterized by FACS. Thismodified ELISPOT approach proved extremely sensitive since backgroundresponses from control wells were low (7.2±0.2 IFNγ-producing SFC/10⁶splenocytes in H-2b and 9.2±0.5 in H-2d mice). Results as shown in FIG.4A revealed that vaccination with pMARV induced 9 measurable epitopes inH-2^(b) mice and 11 in H-2^(d), pEBOS induced 9 and 8, and pEBOZgenerated 10 and 12, in these respective strains. While five of nine(55.6%) of the epitopes from pMARV-immunized H-2^(b) mice were CD8+,they accounted for about 57.3% of the total MGP-specific IFNγ+ responseas measured by both ELISPOT and FACS confirmation and phenotypicanalysis. Similarly, only 33% and 38% of confirmed epitopes wereCD8-restricted in pEBOS-immunized H-2^(b) and H-2^(d) mice,respectively. However these epitopes comprised roughly 50-90% of thetotal response; CD8+ T cell responses were estimated to be approximately56% in both mouse strains while FACS estimates were 51% and 90% inH-2^(b) and H-2^(d) mice, respectively. Total CD8+ responses were lowerin pEBOZ-vaccinated animals and measured between 33% and 57% (33% forboth strains by ELISPOT and 6% and 57% for H-2^(b) and H-2^(d) mice,respectively, by FACS).

A single immunodominant epitope was detected in both mouse strainsreceiving pEBOS where an immunodominant epitope was loosely defined asgenerating an IFNγ response at least two-fold over the highestsubdominant epitope; pMARV induced four H-2^(b)-restrictedimmunodominant CD8+ epitopes within peptides MGP₂₅₋₃₉ (#5), MGP₆₇₋₈₁(#12), MGP₁₈₁₋₁₉₅ (#31) and MGP₃₈₅₋₃₉₉ (#65), and an H-2^(d)-restrictedCD4+ epitope in MGP₁₅₁₋₁₇₁ (#27). Four of these epitopes occurred withinhighly conserved regions of MARV GP1, including three of which werelocated within the putative receptor binding domain, while only oneoccurred within the variable mucin-like region (MGP₃₈₅₋₃₉₉ (#65)) asshown in FIG. 4B and FIG. 4C. pEBOS stimulated CD8+ epitopes occurringin SUDV GP (SGP)₁₉-33 (#4) and SGP₂₄₁₋₂₅₅ (#41) in H-2b and H-2^(d)mice, respectively, both in highly conserved regions of GP1. However,pEBOZ immunization revealed three immunodominant epitopes in H-2^(d)mice (a CD8-restricted epitope located in the ZEBOV GP receptor bindingdomain (GP)₁₃₉₋₁₅₃ (#24), and two CD4-restricted epitopes ZGP₁₇₅₋₁₈₉(#30) and ZGP₃₉₁₋₄₀₅ (#66)), occurring within the receptor bindingdomain and the mucin-like region, respectively. Only one immunodominantepitope was defined in H-2^(b) mice which contained both a CD4+ and aCD8+ epitope (#89) and occurred in a highly conserved region of GP2.Overall, diverse epitope hierarchies were consistent and reproducible ineach vaccine group. Furthermore, as shown in FIG. 4D, the subdominantresponse comprised a significant proportion of the total response; thetotal AVE subdominant response as measured by the modified ELISPOT assaywas approximately 12%, 62%, and 74% in pMARV-, pEBOS- andpEBOZ-immunized H-2^(b) mice, respectively, while responses in H-2^(d)mice were 47%, 50% and 34%, respectively.

Lastly, total GP-specific T cell responses were measured by FACS usingstimulation with minimal peptide pools containing only confirmedepitope-comprising peptides identified. . Robust responses were detectedin each of the vaccinated animals and were, in a majority of cases,comprised by both activated CD4+ and CD8+ T cells. Responses wereGP-specific, since little IFNγ production was observed with a controlpeptide (h-Clip), and correlated well with ELISPOT data. The onlyinstance where immunization did not induce remarkable CTL as measured byFACS was in H-2^(d) mice vaccinated with pMARV in which no epitopeidentified by ELISPOT was confirmed to be CD8-restricted. Altogether,these data show that each of the vaccine plasmids was highly immunogenicin mice and yielded robust GP-specific T cell responses recognizing adiverse array of T cell epitopes including immunodominant epitopeswithin highly conserved regions of the GP. Furthermore, the highlydiverse subdominant T cell response characterized herein might haveotherwise been overlooked using traditional matrix array peptide poolsfor epitope identification.

T cell responses were measured for reactivity against minimal peptidepools comprised by all positively identified peptides for eachrespective GP by FACS. FIG. 7A shows DNA vaccine-induced T cellresponses are shown from a representative animal and IFNγ-producing CD4+(right) and CD8+ (left) cells are gated. FACS plots are shown.Incubation with h-CLIP peptide served as a negative control (Control).FIG. 7B shows results of gated cells in FIG. 7A are summarized asaverage % of total CD44+/IFNγ+ CD4+ or CD8+ cells and error barsrepresent SEM. Experiments were repeated at least two times with similarresults.

‘Single-Dose’ Protection in Mice

Vaccine efficacy against ZEBOV challenge was next assessed in thepreclinical murine model. Mice were vaccinated only once due to strongNAb induction and protection data observed. Mice (H-2^(k); n=10/group)were immunized with 40 μg of the pEBOZ DNA and protection was evaluated28 days later by challenge with 1,000 LD₅₀ of mouse-adapted ZEBOV(mZEBOV) in a BSL4 facility. While all control animals succumbed toinfection by day 7 post-challenge, FIG. 5A shows DNA-vaccinated micewere completely protected (P=0.0002). In addition, as shown in FIG. 5B,control mice exhibited progressive loss of body weight until death(P<0.0001).

To better understand the mechanisms of DNA-induced protection in a‘single-dose’ model, we next assessed NAb and T cell generation. NAbswere assessed 25 days post-vaccination, 3 days prior to challenge, and,as shown in FIG. 5C, a significant (P<0.0001) increase was detected inall vaccinated animals (n=10/group); reciprocal endpoint dilution titersranged from 19 to 42, 27.3±2.5.

We next evaluated the generation of ZGP-specific T cells and increasedthe scope of our analysis to compare responses in mice immunized witheither the pEBOZ alone, or in a trivalent formulation IFN-γ production(n=5) was assessed 11 days later by FACS using whole ZGP peptide pools;the data is shown in FIG. 5D. IFNγ-producing T cells were detected inall animals and were specific for ZGP peptides since stimulation with acontrol peptide did not induce cytokine production. Immunization witheither the monovalent or trivalent formulation induced robust IFNγ Tcell responses that, when compared, were not significantly differentP=0.0920).

Since CTL may be important in eliminating virus-infected cells (WarfieldKL, et al. (2005). Induction of humoral and CD8+ T cell responses arerequired for protection against lethal Ebola virus infection. J Immunol175: 1184-1191; Kalina W V, Warfield K L, Olinger G G, Bavari S (2009).Discovery of common Marburgvirus protective epitopes in a BALB/c mousemodel. Virol J 6: 132; Olinger G G, et al. (2005). Protective cytotoxicT-cell responses induced by venezuelan equine encephalitis virusreplicons expressing Ebola virus proteins. J Virol 79: 14189-14196;Sullivan N J, et al. (2011). CD8(+) cellular immunity mediates rAd5vaccine protection against Ebola virus infection of nonhuman primates.Nat Med 17: 1128-1131; and Geisbert T W, et al. (2010). Vector choicedetermines immunogenicity and potency of genetic vaccines against AngolaMarburg virus in nonhuman primates. J Virol 84: 10386-10394), productionof an additional effector cytokine, TNF, as well as a developmentalrestriction factor, T-box transcription factor TBX21 (T-bet), known tocorrelate with T_(h)1-type CTL immunity and cytotoxicity were measuredand the results were as follows. For Total Cells: TNF 2.9±0.8, Tbet13.0±1.1. For CD4+/CD44+/IFNγ+ Cells: TNF 61.4±3.1, Tbet 72.6±2.0. ForCD8+/CD44+/IFNγ+ Cells: TNF 33.0±3.3, Tbet 992.1±1.4 (*p<0.1;***p<0.001; *** p<0.0001). We found that ˜61% and ˜33% of activated CD4+and CD8+ T cells, respectively, also produced TNF in addition to IFNγ.Furthermore, a majority of IFNγ-producing T cells expressed high levelsof T-bet; about 73% and 92% of CD8+ and CD4+ T cells, respectively, wereCD44+ and produced IFNγ following ZGP peptide stimulation.

FIGS. 8A and 8B show T cell induction by ‘single-dose’ vaccination. Tcell responses in H-2^(k) mice after a single pEBOZ immunization or asingle trivalent vaccination, comprised by the three vaccine plasmids inseparate sites, as measured by FACS are shown (a) and summarized (b) asAVE % of total CD44+/IFNγ+ CD4+ (purple) or CD8+ (orange) cells.Pseudocolor FACS plots are from a representative animal andIFNγ-producing CD4+ (right) and CD8+ (left) cells are gated. Incubationwith h-CLIP peptide served as a negative control (Control). Experimentswere performed twice with similar results, error bars represent SEM; ns,no significance.

Discussion

We report development and evaluation of a polyvalent-filoviral vaccinein preclinical rodent immunogenicity and efficacy studies. Completeprotection against challenge with gpMARV and gpZEBOV was observedfollowing two DNA vaccine doses in guinea pigs, as well as with a‘single-dose’ DNA vaccine in mice against mZEBOV. To date, geneticvaccination of guinea pigs has included either injection of naked DNA(Sullivan N J, Sanchez A, Rollin P E, Yang Z Y, Nabel G J (2000).Development of a preventive vaccine for Ebola virus infection inprimates. Nature 408: 605-609) or DNA delivered by gene gun (Dowling W,et al. (2006). The influences of glycosylation on the antigenicity,immunogenicity, and protective efficacy of Ebola virus GP DNA vaccines.J Virol 81: 1821-1837; Vanderzanden L, et al. (1998). DNA vaccinesexpressing either the GP or NP genes of Ebola virus protect mice fromlethal challenge. Virology 246: 134-144; and Riemenschneider J, et al(2003). Comparison of individual and combination DNA vaccines for B.anthracis, Ebola virus, Marburg virus and Venezuelan equine encephalitisvirus. Vaccine 21: 4071-4080), however, either method required at leastthree vaccinations to achieve complete protection. Improved protectionherein may be due to the induction of robust Abs since a single DNAvaccination generated GP-specific IgG binder titers that were comparablein magnitude to titers in protected animals following gene gunadministration; DNA vaccination induced 3.85 and 2.18 log 10 ZGP andMGP-specific Ab titers, respectively, after a single administrationversus 2.7 and 3.0 after three gene gun vaccinations. For comparisonwith an alternative ‘single-dose’ protective strategy in guinea pigs, anAg-coupled virus-like particle (VLP) platform generated Ab titers thatwere only slightly higher than observed following DNA vaccination(Swenson D L, Warfield K L, Negley D L, Schmaljohn A, Aman M J, Bavari S(2005). Virus-like particles exhibit potential as a pan-filovirusvaccine for both Ebola and Marburg viral infections. Vaccine 23:3033-3042). Furthermore, a recombinant adenovirus (rAd) approach inducedZGP-specific NAb titers that were lower than those from a single DNAvaccination (53 reciprocal endpoint dilution titer verses 88 herein)(Kobinger G P, et al. (2006). Chimpanzee adenovirus vaccine protectsagainst Zaire Ebola virus. Virology 346: 394-401). Vaccination with rVSV(Jones S M, et al (2007). Assessment of a vesicular stomatitisvirus-based vaccine by use of the mouse model of Ebola virus hemorrhagicfever. J Infect Dis 196 Suppl 2: S404-412) generated ZGP-specific Abtiters that were similar to the current platform. Altogether, these datademonstrate that DNA vaccination was capable of inducing binding andneutralizing Abs that were comparable to non-replicating viral platformsand that these data may help, in part, to explain strong guinea pigsurvival data herein.

The generation of NAbs by protective DNA vaccination may have benefitedby transgene-expressed mature GP structures. In vitro transfectionstudies confirmed that the vaccine-encoded GP were highly expressed,post-translationally cleaved (FIG. 1B), transported to the cell surface,and sterically occluded the immunodetection of cell surface molecules(FIG. 1C). Therefore, it was highly likely that the vaccine immunogensformed herein matured into hetero-trimeric spikes that would otherwisebe functional upon virion assembly during infection. This may beimportant for the generation and display of virologically-relevantneutralizing determinants which would be subsequently critical for theinduction of conformation-dependent Nabs (Dowling W, et al. (2007).Influences of glycosylation on antigenicity, immunogenicity, andprotective efficacy of ebola virus GP DNA vaccines. J Virol 81:1821-1837; Shedlock D J, Bailey M A, Popernack P M, Cunningham J M,Burton D R, Sullivan N J (2010). Antibody-mediated neutralization ofEbola virus can occur by two distinct mechanisms. Virology 401:228-235). Thus, in this regard, the expression of native anchoredstructures may be superior to soluble derivatives in the capacity forgenerating NAbs (Sullivan N J, et al. (2006). Immune protection ofnonhuman primates against Ebola virus with single low-dose adenovirusvectors encoding modified GPs. PLoS Med 3: e177; Xu L, et al. (1998).Immunization for Ebola virus infection. Nat Med 4: 37-42).

To better characterize T cells responses as driven by a protectivevaccine, we performed immunogenicity and efficacy studies in mice anddetermined ‘single-dose’ complete protection against mZEBOV with DNAvaccination (FIGS. 5A-5D). To date, the most effective platformsconferring complete protection in this model are VLP, either with(Warfield K L, et al. (2005). Induction of humoral and CD8+ T cellresponses are required for protection against lethal Ebola virusinfection. J Immunol 175: 1184-1191; Warfield K L, Swenson D L, OlingerG G, Kalina W V, Aman M J, Bavari S (2007). Ebola virus-likeparticle-based vaccine protects nonhuman primates against lethal Ebolavirus challenge. J Infect Dis 196 Suppl 2: S430-437) or without (Sun Y,et al. (2009). Protection against lethal challenge by Ebola virus-likeparticles produced in insect cells. Virology 383: 12-21) adjuvant, rAdvaccination ((Kobinger G P, et al. (2006) SUPRA; Choi J H, et al.(2012). A single sublingual dose of an adenovirus-based vaccine protectsagainst lethal Ebola challenge in mice and guinea pigs. Mol Pharm 9:156-167; Richardson J S, et al. (2009). Enhanced protection againstEbola virus mediated by an improved adenovirus-based vaccine. PLoS One4: e5308), or rRABV vaccination (Blaney J E, et al. (2011). Inactivatedor live-attenuated bivalent vaccines that confer protection againstrabies and Ebola viruses. J Virol 85: 10605-10616). However,characterization of T cell responses were severely limited in thesestudies and were restricted to splenocyte stimulation with either two(Warfield K L, (2007), SUPRA) or one (Warfield K L, et al. (2005) SUPRA)peptides previously described to contain ZGP T cell epitopes (Warfield KL, et al. (2005) SUPRA. Olinger G G, et al. (2005) SUPRA; Kobinger G P,et al. (2006), SUPRA; Sun Y, et al. (2009). Choi, J H, et al. (2012).Herein, we report induction of robust and broad CTL by protectivevaccination as extensively analyzed by a novel modified T cell assay(FIG. 4A and Tables 1-6). In total, 52 novel T cell epitopes wereidentified including numerous immunodominant epitopes occurringprimarily in highly conserved regions of GP. Of the 22 total ZGPepitopes identified, only 4 have been previously reported. Moreover,only one of the 20 MGP (Kalina W V, Warfield K L, Olinger G G, Bavari S(2009). Discovery of common Marburgvirus protective epitopes in a BALB/cmouse model. Virol J 6: 132) and one of 16 SGP epitopes were previouslydescribed. As such, this the most comprehensive report of preclinical GPepitopes to date, describing GP epitopes from multiple filoviruses intwo different mouse genetic backgrounds.

Another novel finding resulting from these analyses was the assessmentof the vaccine-induced subdominant T cell responses, which we showcomprised a significant percentage of the total T cell response, widelyranging between 12%-74% (FIG. 4D). This may be particularly importantsince subdominant responses can significantly contribute to protection.Thus, it may prove informative in the future to determine the specificcontributions of the subdominant and immunodominant epitopic T cellresponses to protection. Notably, these responses may have otherwisebeen overlooked using traditional matrix array peptide pools for epitopeidentification. As such, limited epitope detection in previous studiesmay have been directly related to lower levels of vaccine-inducedimmunity, the use of less sensitive standard assays, and/or the use ofpeptide arrangements and/or algorithms favoring detection ofimmunodominant CD8+ epitopes.

Although immune correlates of protection against the filoviruses remaincontroversial, data generated by this highly immunogenic approachprovides a unique opportunity with which to study T cell immunity asdriven by a protective vaccine. DNA vaccination herein induced strongZGP-specific T cells, a large part of which were characterized byT_(h)1-type multifunctional CTL expressing high levels of T-bet , alsoshown to correlate with T cell cytotoxicity in humans. It is clear thatprevious stand-alone DNA vaccine platforms capable of generating mainlyhumoral immune responses and cellular immunity skewed towards CD4+ Tcells may likely benefit from in vivo EP delivery which has beenrecently demonstrated to induce potent CD8+ T cells in NHPs and theclinic. Thus, data herein are consistent with this approach as astand-alone or prime-boost modality in NHP immunogenicity and efficacystudies. This approach offers an attractive vaccination strategy thatcan be quickly and inexpensively modified and/or produced for rapidresponse during Filoviridae bio-threat situations and outbreaks. Inaddition, this model approach provides an important tool for studyingprotective immune correlates against filoviral disease and could beapplied to existing platforms to guide future strategies.

Example 2

A trivalent vaccine is provided which comprises three plasmids. Thefirst plasmid comprises a nucleic acid sequence that encodes a Zaireebolavirus consensus immunogen which is based upon ZEBOV CON, SEQ IDNO:1, modified to include an IgE signal peptide at the N terminus of theZaire ebolavirus consensus immunogen. The second plasmid comprises anucleic acid sequence that encodes a Sudan ebolavirus consensusimmunogen which is based upon SUDV CON, SEQ ID NO:2, modified to includean IgE signal peptide at the N terminus of the Sudan ebolavirusconsensus immunogen. The third plasmid comprises a nucleic acid sequencethat encodes a Marburg marburgvirus Angola (MARV immunogen which isbased upon MARV ANG, SEQ ID NO:3, modified to include an IgE signalpeptide at the N terminus of the Marburg marburgvirus Angola immunogen.

Example 3

A five plasmid vaccine is provided. The first plasmid comprises anucleic acid sequence that encodes a Zaire ebolavirus consensusimmunogen which is ZEBOV CON, SEQ ID NO:1. The second plasmid comprisesa nucleic acid sequence that encodes a Sudan ebolavirus consensusimmunogen which is SUDV CON, SEQ ID NO:2. The third plasmid comprises anucleic acid sequence that encodes SEQ IDNO:4, a Marburgmarburgvirus-Ravn cluster consensus (MARV-RAV CON) using Marburgmarburgvirus Ravn, Durba (09DRC99) and Uganda (02Uga07Y). The fourthplasmid comprises a nucleic acid sequence that encodes SEQ IDNO:5, aMarburg marburgvirus-Ozolin cluster consensus (MARV-OZO CON) usingOzolin, Uganda (01Uga07), and Durba (05 and 07DRC99). The fifth plasmidcomprises a nucleic acid sequence that encodes SEQ IDNO:6, a Marburgmarburgvirus-Musoke cluster consensus (MARV-MUS CON) using (Musoke,Popp, and Leiden).

Example 4

A five plasmid vaccine is provided. The first plasmid comprises anucleic acid sequence that encodes a Zaire ebolavirus consensusimmunogen which is based upon ZEBOV CON, SEQ ID NO:1, modified toinclude an IgE signal peptide at the N terminus of the Zaire ebolavirusconsensus immunogen. The second plasmid comprises a nucleic acidsequence that encodes a Sudan ebolavirus consensus immunogen which isbased upon SUDV CON, SEQ ID NO:2, modified to include an IgE signalpeptide at the N terminus of the Sudan ebolavirus consensus immunogen.The third plasmid comprises a nucleic acid sequence that encodes Marburgmarburgvirus Rav consensus based upon SEQ IDNO:4, a Marburgmarburgvirus-Ravn cluster consensus (MARV-RAV CON) using Marburgmarburgvirus Ravn Durba (09DRC99) and Uganda (02Uga07Y) and modified toinclude an IgE signal peptide at the N terminus of the consensus Marburgmarburgvirus-Rav immunogen. The fourth plasmid comprises a nucleic acidsequence that encodes Marburg marburgvirus Ozo consensus based upon SEQIDNO:5, a Marburg marburgvirus-Ozolin cluster consensus (MARV-OZO CON)using Ozolin,Uganda (01Uga07), and Durba (05 and 07DRC99) and modifiedto include an IgE signal peptide at the N terminus of the consensusMarburg marburgvirus-Ozo immunogen. The fifth plasmid comprises anucleic acid sequence that encodes Marburg marburgvirus Mus consensusbased upon SEQ IDNO:6, a Marburg marburgvirus-Musoke cluster consensus(MARV-MUS CON) using (Musoke, Popp, and Leiden) and modified to includean IgE signal peptide at the N terminus of the consensus Marburgmarburgvirus-Mus immunogen.

Example 5

A six plasmid vaccine is provided. The first plasmid comprises a nucleicacid sequence that encodes a Zaire ebolavirus consensus immunogen whichis ZEBOV CON, SEQ ID NO:1. The second plasmid comprises a nucleic acidsequence that encodes a Sudan ebolavirus consensus immunogen which isSUDV CON, SEQ ID NO:2. The third plasmid comprises a nucleic acidsequence that encodes SEQ IDNO:4, a Marburg marburgvirus-Ravn clusterconsensus (MARV-RAV CON) using Marburg marburgvirus-Ravn, Durba(09DRC99) and Uganda (02Uga07Y). The fourth plasmid comprises a nucleicacid sequence that encodes SEQ IDNO:5, a Marburg marburgvirus-Ozolincluster consensus (MARV-OZO CON) using Ozolin, Uganda (O1Uga07), andDurba (05 and 07DRC99). The fifth plasmid comprises a nucleic acidsequence that encodes SEQ IDNO:6, a Marburg marburgvirus-Musoke clusterconsensus (MARV-MUS CON) using (Musoke, Popp, and Leiden). The sixthplasmid comprises a nucleic acid sequence that encodes SEQ IDNO:3, aMarburg marburgvirus Angola 2005 isolate glycoproteins immunogen.

Example 6

A five plasmid vaccine is provided. The first plasmid comprises anucleic acid sequence that encodes a Zaire ebolavirus consensusimmunogen which is based upon ZEBOV CON, SEQ ID NO:1, modified toinclude an IgE signal peptide at the N terminus of the Zaire ebolavirusconsensus immunogen. The second plasmid comprises a nucleic acidsequence that encodes a Sudan ebolavirus consensus immunogen which isbased upon SUDV CON, SEQ ID NO:2, modified to include an IgE signalpeptide at the N terminus of the Sudan ebolavirus consensus immunogen.The third plasmid comprises a nucleic acid sequence that encodes Marburgmarburgvirus Rav consensus based upon SEQ IDNO:4, a Marburgmarburgvirus-Ravn cluster consensus (MARV-RAV CON) using Marburgmarburgvirus Ravn Durba (09DRC99) and Uganda (02Uga07Y) and modified toinclude an IgE signal peptide at the N terminus of the consensus Marburgmarburgvirus-Rav immunogen. The fourth plasmid comprises a nucleic acidsequence that encodes Marburg marburgvirus Ozo consensus based upon SEQIDNO:5, a Marburg marburgvirus-Ozolin cluster consensus (MARV-OZO CON)using Ozolin,Uganda (01Uga07), and Durba (05 and 07DRC99) and modifiedto include an IgE signal peptide at the N terminus of the consensusMarburg marburgvirus-Ozo immunogen. The fifth plasmid comprises anucleic acid sequence that encodes Marburg marburgvirus Mus consensusbased upon SEQ IDNO:6, a Marburg marburgvirus-Musoke cluster consensus(MARV-MUS CON) using (Musoke, Popp, and Leiden) and modified to includean IgE signal peptide at the N terminus of the consensus Marburgmarburgvirus-Mus immunogen. The sixth plasmid comprises a nucleic acidsequence that encodes a Marburg marburgvirus Angola 2005 isolateglycoproteins immunogen which is based upon MARV ANG, SEQ ID NO:3,modified to include an IgE signal peptide at the N terminus of theMarburg marburgvirus Angola immunogen.

Example 7

Described herein is a DNA vaccine formulation expressing 3 syntheticZaire Ebola virus (EBOV) glycoproteins (GP): 2 designed based on GPsequence alignments (1976-2014) and a 3rd construct matched to a 2014outbreak strain. Plasmid IL-12 (pIL-12) was also included as an adjuvantto further enhance cellular immune responses. The multivalent GP DNAvaccine formulation was administered in macaques following a DNA-DNAprime-boost immunization regimen. Macaques (n=3 or 4/group) received themultivalent GP DNA formulation +pIL-12 by intramuscular deliveryfollowed by electroporation. Differences in immunogenicity were assayedand protection between different doses, regimens (2, 3, 4, and 5injections), and different spacing intervals between subsequent doseswere monitored. Both antibody and T cell responses were observed in 83%of animals 2 weeks following the first injection and 100% of animalsafter the 2nd injection. The macaques were challenged with a lethal doseof the EBOV Guinea-Makona outbreak strain (1000 pfu, 7-U virus) andmonitored for 28 days following infection. 100% of animals receiving atleast 3 injections at 4 week intervals survived lethal challenge.Animals were fully protected against signs of disease and did notexhibit elevated blood chemistry. Interestingly, 50% of animalsreceiving 2 injections survived lethal challenge. The surviving animalsexhibited minimal signs of disease, suggesting that with furtheroptimization complete protection with 2 injections is potentiallyachievable. In additional optimization studies in mice, singleinjections were found to be 100% protective and long-term immuneresponses 8 months post vaccination were induced.

Methods Developing an EBOV GP DNA Vaccine Formulation

Vaccines currently in clinical trials include rVSVAG/ZEBOVGP, ChAd3prime+MVA boost, and MVA pan-Filovirus. While these vaccines areimmunogenic, protective in non-human primates (NHPs), and provide singledose protection, they develop anti-vector immunity, have uncertainduration of memory response, give adverse reactions in human clinicaltrials and may not be suitable for all populations. Thus, an additionalplatform with a cleaner safety profile that can induce strong immuneresponses against heterologous Zaire Ebola viruses would be verybeneficial (FIG. 9).

Three EBOV DNA constructs were designed: a consensus sequence of ZEBOV(1976-1996) (ConEBOVGP #1), ZEBOV (2002-2008) (ConEBOVGP #2), and amatched ZEBOV sequence from the 2014 Guinea outbreak. (Guinea-GP). Fivevaccines were developed, monovalent vaccines which comprise only asingle DNA construct, a bivalent vaccine formulation which compriseeither ConEBOVGP #1 with Guinea-GP and a trivalent formulation whichcomprises all three of ConEBOVGP #1, ConEBOVGP #2, and Guinea-GP (FIG.10).

Results A Single Immunization of DNA Vaccine is Immunogenic in Mice

BALB/c mice received a single intramusclular (IM) immunization followedby electroporation (EP) of the vaccine formulation. On day 28, Total IgGantibody titer and ELISPOT-IFNγ were determined. Each vaccineformulation produced a robust IgG response. Polyfunctional CD4+ and CD8+T cells secreting IFNγ, IL2, and TNFα were similar for all vaccines andformulations. (FIG. 11).

A Single Immunization is Fully Protective in Mice Against LethalMouse-Adapted Ebola Virus Challenge

BALB/c mice immunized with bivalent or trivalent vaccine formulationswere lethal challenged with 1000LD₅₀ of heterologous Ebolavirus. Thechallenge strain was mouse adapted-Ebola Mayinga 1976. Mice vaccinatedwith the control plasmid, pVax1, quickly lost weight and did not survivepast day 7. However, both the mice vaccinated with the bivalent and micevaccinated with the trivalent vaccine formulations maintained theirweight and had no deceased mice up to day 20 (FIG. 12).

Individual GP DNA Vaccine Constructs Induce Robust Memory Responses inMice.

BALB/c mice received a 3×IM 40 μg immunization of a monovalent vaccinefollowed by electroporation on days 0, 28 and 84. IgG antibody titer,INFγ, CD4+CD44+ memory T cells, and CD8+CD44+ memory T cells weremeasured. Robust immune responses were detectable months after lastinjection (FIG. 13).

GP DNA Vaccine Formulations are Immunogenic in NHPs

Cynomolgus macaques were used as a NHP because they are a model forEbola vaccine efficacy and lethal challenge. Macques were administeredeither a bivalent formulation with a Rhesus pIL12 adjuvant or atrivalent formulation with a Rhesus pIL12 adjuvant I.M. followed by EP.Different injection regimens in order to understand immunogenicity.Group 1 received 2 IM-EP injections of the bivalent formulation at a 4week interval. Group 2 received 2 IM-EP injections of the trivalentformulation at a 4 week interval. Group 3 received 3 IM-EP injections ofthe trivalent formulation. Samples for immunogenicity studies were takenmonthly and for one additional month following the last dose. (FIG. 6).Each DNA vaccine formulation induced robust anti-Ebola GP antibodyresponses (GMT>10³) & anti-GP T-cell responses. The immune responseswere boosted following each injection (FIG. 14).

GP DNA Formulation Vaccines Protect Against Lethal Zaire Ebola Virus(Makona) Challenge

The Cynomolgus macaques from groups 1, 2 and 3 were challenged with a1000 TCID50 Guinea-Makona 2014 C07 virus (7-U reference strain) dose 28days post-final DNA immunization. Animals were monitored for 28 dayspost-challenge. While none of the control animals survived past day 10post challenge, 4/4 group 3 animals survived 28 days post challenge, 4/8group 2 animals survived 28 days post challenge and 3/4 group 1 animalssurvived 28 days post challenge (FIG. 15). Surviving animals did nothave any significant signs of disease. They also maintained normal CBCand enzyme levels. Bivalent and Trivalent DNA vaccines delivered byIM-EP elicit long-term antibody and T cell responses that weredetectable >3 months post final DNA injection (FIG. 43).

Three doses of a Trivalent EBOV GP DNA vaccine is 100% protectiveagainst lethal EBOV challenge. Two doses of a Bivalent EBOV GP DNAvaccine affords 75% protection. Overall, the data supports further studyof DNA vaccines, delivered by IM-EP, for possible administration againstEbola and other infectious pathogens

EBOV-001 Phase I Clinical

An Open-Label study of INO-4212 (with or without INO-9012 was conducted.INO-4212 was administered IM or ID followed by electroporation inhealthy volunteers. Safety and immunological assessments were monitored.Intradermal delivery and intramuscular delivery were compared. Therewere 69 total subjects. ELISA analysis was performed before immunization(baseline), and at weeks 2, 6, and 14. Seriopositive is defined as apositive IgG antibody response to Ebola Zaire glycoprotein.

INO-4201 is a DNA vaccine formulated with the consensus envelopeglycoprotein of Zaire Ebolavirus (ConEBOVGP #1) generated by using theenvelope glycoprotein sequences of the 1976, 1994, 1995, 1996, 2003,2005, 2007 and 2008 outbreak strains, driven by a human CMV promoter(hCMV promoter) with the bovine growth hormone 3′end poly-adenylationsignal (bGH polyA). pGX4201 was made by cloning the synthetic consensusenvelope glycoprotein gene of Zaire Ebolavirus into pGX0001 at the BamHIand XhoI sites.

The ConEBOVGP #1 (ConGP1) sequence was constructed by generating aconsensus envelope glycoprotein sequence of Zaire Ebolavirus using theenvelope glycoprotein sequences of the 1976, 1994, 1995, 1996, 2003,2005, 2007 and 2008 outbreak strains. Briefly, a consensus GP sequencewas first generated based on six envelope sequences of the 1976, 1994,1995, 1996, 2003 and 2005 outbreak strains. Then three non-consensusresidues at the positions 377, 430 and 440 were weighted towards the2003, 2005, 2007 and 2008 strains since they were the most recent andlethal outbreaks with published sequence data. The GenBank accessionnumbers for selected outbreak strain GP sequences are: Q05320, P87671,AAC57989, AEK25495, ABW34743, P87666, AER59718, AER59712, ABW34742,AAL25818. Once the consensus GP1 sequence was obtained, an upstreamKozak sequence was added to the N-terminal. Furthermore, in order tohave a higher level of expression, the codon usage of this gene wasadapted to the codon bias of Homo sapiens genes. In addition, RNAoptimization was also performed: regions of very high (>80%) or very low(<30%) GC content and the cis-acting sequence motifs such as internalTATA boxes, chi-sites and ribosomal entry sites were avoided. Thesynthesized ConGP1 was digested with BamHI and XhoI, and cloned into theexpression vector.

INO-4202 is a DNA vaccine formulated with a DNA plasmid expressing theenvelope glycoprotein of Zaire Ebolavirus isolated from the 2014outbreak in Guinea (GuineaGP), driven by a human CMV promoter (hCMVpromoter) with the bovine growth hormone 3′end poly-adenylation signal(bGH polyA).

INO-4212 is a bivalent vaccine of INO-4201 and INO-4202.

FIG. 17 depicts the vaccine formulation schedule, route and dose foreach cohort. After the first injection, 15% or less of the patients wasseriopositive. After the second injection, 50-100% of the patients wereseriopositive. After the third injection 79-100% of the patients wereseriopositive (FIG. 18). Two representative patients with a moderateIFNγ ELISpot, or a high IFNγ ELISpot showed specific T-cell responses(FIG. 19).

While other Ebola vaccines platforms, including NIAID VRC/GSK andrVSV/ZEBOVGP, are currently in clinical trials the present bivalent andtrivalent vaccines described herein have advantages not observed in theother vaccine platforms. For example, the bi- or tri-valent vaccines canbe administered IM or ID, while the other vaccine platforms are onlyadministered IM. Importantly, NIAID VRC/GSK and rVSV/ZEBOVGP show sideeffects including fever, fatigue, arthralgia, and lymphopenia while thebi- and tri-valent vaccines do not show any side effects. It should benoted however, that some of the side effects of rVSV/ZEBOVGP andChAd3/MVAGP overlap with symptoms of Ebola. Further the bi- andtri-valent vaccines give antibody titers one to two orders of magnitudelarger than rVSV/ZEBOVGP and ChAd3/MVAGP (FIG. 20).

Subjects (n=15) were assigned to receive INO-4201 at a 2 mg DNA/dosegiven as two separate 1 mg (0.1 mL) ID (Mantoux) injections followed byEP with the CELLECTRA®-3P device. Subjects received a 3-dose series withimmunizations at 0, 4 weeks, and 12 weeks (0-4-12 week schedule).Antibodies specific for EBOV glycoprotein (GP) were measured from thesera of vaccinated subjects with a binding ELISA. Reciprocal endpointtiters above Day 0 are shown two weeks post each immunization (FIG. 22).100% of subjects vaccinated with INO-4201 seroconverted after 2immunizations (FIG. 22, Cohort 3 and FIG. 24).

Example 8

Described herein is immune response data for the three EBOV DNAconstructs described in Example 7: a consensus sequence of ZEBOV(1976-1996) (ConEBOVGP #1 or INO-4201), ZEBOV (2002-2008) (ConEBOVGP#2), and a matched ZEBOV sequence from the 2014 Guinea outbreak.(Guinea-GP). Five vaccines were developed, monovalent vaccines whichcomprise only a single DNA construct, a bivalent vaccine formulationwhich comprise either ConEBOVGP #1 with Guinea-GP and a trivalentformulation which comprises all three of ConEBOVGP #1, ConEBOVGP #2, andGuinea-GP. 69 Subjects from cohorts 1-5 were included in analysis ofimmune response by ELISA and 75 subjects from cohorts 1-5 were includedin analysis of immune response by ELISpot (FIGS. 21, 25).

ELISA Titers by Cohort and Timepoint

Titers of anti-EBOVR were determined for each cohort at weeks 2, 6 and14. By week 6, each cohort saw an increase in antibody titer above Day 0(FIGS. 22-23). There was little to no reactivity for the first dose ineach cohort. Dose 2 begins to drive seroconversion, with Cohorts 3 and 5seeing the largest frequency. Dose three drives >90% serconversion in4/5 cohorts. Cohorts 3 and 5 show 100% seroconversion at this time point(FIG. 24)

Subject Responses by Peptide Pool

Cohort responses were analyzed by peptide pool (FIGS. 26-33). To analyzeELISpot outliers, day 0 values for each pool and total EBOV responseswere used to create an outlier threshold (Mean day 0 values +(3× STDEVof day 0 values)). This threshold should encompass 99% of a normallydistributed population. Any subject that displayed baseline valuesgreater than the outlier threshold was removed and a responder criteriawas generated with the remaining subjects.

ICS Analysis

47 subjects from all cohorts were included in the analysis, howevercohort 5 is underrepresented (FIG. 35). ICS analysis performed atbaseline and week 14. A single EBOV peptide pool composed of Pools 1-4is used for stimulation. Analysis of T cell activity in the form of IFNgor TNFα production from both CD4 and CD8 compartments suggestssignificant elevation of TNFα in both CD4 and CD8 compartments as wellas elevation of TNFα and/or IFNg in Cohort 3 only (Wilcoxon matchedpaired analysis, two-tailed)

Immunology Summary

100% of Cohort 3 (ID) patients seroconverted after 2 doses. 92% ofCohort 5 (IM+IL12) patients seroconverted after 2 doses and 100% after 3doses. Other cohorts showed 67% at best after 2 doses and ranged as highas 93% after 3 doses.

When analyzing all patients: the best response frequency were Cohorts 2and 4 with 53% and 57% respectively. Cohort 3 showed 40% responders.Addition of IL-12 in Cohort 5 did not seem to influence response rates(47%). When analyzing patients with 8 outliers removed the responsefrequency were Cohorts 2 and 4 with 84.6% and 76.9% respectively. Cohort3 showed 64.3% responders. Addition of IL-12 in Cohort 5 did not seem toinfluence response rates (53.3%).

Both CD4 and CD8 T cells showed high expression of TNFα and TNFα and/orIFNg in Cohort 3 (statistically significant to baseline, Wilcoxonmatched pairs test, 2 tailed).

Immunization with INO-4201 was well tolerated in healthy volunteers withno Grade 3 or Grade 4 SAEs noted. INO-4201 induced robust EbolaGP-specific antibody (GMT 46,968) and resulted in 100% seroconversion,as gauged by binding ELISA, after only two doses of INO-4201.Administration of INO-4201 generated EBOV GP specific T cell responsesas assessed by Interferon gamma (IFNγ) ELISpot (295.3 SFU per 10⁶ PBMCs)and significant increases in in the production of IFNγ or TNFα in boththe CD8+ T and CD4+ T cell compartments. Intradermal administration ofINO-4201 using the Cellectra device is both well tolerated andimmunogenic as assessed by both humoral and cellular EBOV GP-specificimmunoassays. These results indicate that INO-4201 is a strong candidatefor further clinical development of a prophylactic Ebola vaccine.

Example 9 Lasting Humoral and Cellular Immune Responses in CynomolgusMacaques Following Administration of a Zaire Ebola Virus (EBOV) GP DNAVaccine Delivered by Intramuscular Electroporation

Presented herein are novel Ebola virus disease (EVD) DNA vaccines thathave a clean safety profile and are serology independent, allowing forpossible repeat vector administration. Three novel synthetic Zaire Ebolavirus (EBOV) GP DNA vaccines were designed and Bivalent, or Trivalentformulations were developed. Both EBOV-GP DNA vaccines were highlyprotective (75-100%) against lethal EBOV Makona C07 challenge incynomolgus macaques. Animals (n=4-5/group) with different regimens werefollowed to monitor long-term immunogenicity following DNA immunization.All NHPs rapidly seroconverted. NHPs have durable total IgG antibodytiters and T cells responses to EBOV GP antigen, includingpolyfunctional CD4 and CD8 T cells expressing IFNγ, IL2, and TNFα andresponses in memory subset populations (FIGS. 47-59). Together, the datastrong support EBOV-GP DNA vaccine delivery for protection and thegeneration of robust memory immune responses.

The EBOV-GP Dan vaccines elicit long-term immune responses and have astrong recall response following a 1 year boost (FIGS. 49-50). Therecall response was remarkably high in the group receiving a single IMinjection (FIG. 50).

Example 10

Presented herein are the peptide sequences and the nucleic acidsequences for the peptides.

TABLE 1 Plasmid Vaccine pMARV GP sequence MARV ANG SEQ FACS Peptide IDELISPOT T cell Number Sequence NO: Position H-2 AVE ±SEM restr. 3IQGVKTLP 7 13-27 d 62 34 4+ ILEIASN 5 ASNIQPQN 8 23-39 b 743 186 8+VDSVCSG 12 SKRWAFRA 9 67-81 b 694 204 4+ GVPPKNV 27 GKVFTEGN 10 137-171d 602 75 4+ IAAM1VN 28 GNIAAMIV 11 163-177 b/d 126 28 8+ NKTVHKMGNIAAMIV 12 d 30 10 4+ NKTVHKM 29 IVNKTVHK 13 169-183 d 92 17 4+ MIFSRQG30 HKMIFSRQ 14 175-189 d 31 10 4+ GQGYRHM 31 RQGQGYRH 15 181-195 b 674112 8+ MNLTSTN 32 RHMNLTST 16 187-201 b 44 16 8+ NKYWTSS 65 LPTENPTT 17385-399 b/ 398/ 107/ 4+ AKSTNST d 16 2 71 PNSTAQHL 18 421-435 d 29 6 4+VYFRRKR 72 HLVYFRRK 19 427-441 d 145 18 4+ RNILWRE 89 GLSWIPFF 20529-543 b 26 8 4+ GPGIEGL 92 GLIKNQNN 21 547-561 d 29 10 4+ LVCRLRR 93NNLVCRLR 22 553-567 d 34 13 4+ RLANQTA 97 TTEERTFS 23 577-591 b 46 18 8+LINRHAI 99 HAIDFLLA 24 589-603 d 63 12 4+ RWGGTCK 101 TCKVLGPD 25601-615 b 97 37 4+ CCIGIED “Epitope-containing peptides were identifiedby IFNγ ELISPOT (≥10 SFC/10⁶ splenocytes AND ≥80% response rate) andthen confirmed by FACS (≥3-5 × 10⁴ CD3+ cells were acquired). Responsesfor each were further characterized by FACS (expression of CD4 and/orCD8 by CD3+/CD44+/IFNγ+ cells). Predicted CD8+ epitopes are underlined(best consensus % rank by IEDB) and previously-described epitopes arereferenced. Immunodominant epitopes are displayed (*).

TABLE 2 Plasmid Vaccine pEBOS GP sequence SUDV CON SEQ FACS Peptide IDELISPOT T cell Number Sequence NO: Position H-2 AVE ±SEM restr. 4FFVWVIIL 26 19-33 b 310 139 8+ FQKAFSM 15 RWGFRSGV 27 85-99 b 108 59 4+PPKVVSY 19 YNLEIKKP 28 109-123 b 55 25 4+ DGSECLP 24 HKAQGTGP 29 139-153d 13 3 8+ CPGDYAF 27 GAFFLYDR 30 157-171 d 29 9 8+ LASTVIY 30 NFAEGVIA31 175-189 d 31 6 4+ FLILAKP 36 SYYATSYL 32 211-225 b 60 16 4+ EYEIENF41 FVLLDRPH 33 241-255 d 338 55 8+ TPQFLFQ 78 NITTAVKT 34 463-477 b/d28/ 12/ 4+ VLPQEST 105 18 82 TGILGSLG 35 487-501 d 82 14 4+ LRKRSRR 83LGLRKRSR 36 493-507 d 69 12 4+ RQVNTRA 89 IAWIPYFG 37 529-543 b 123 408+/ PGAEGIY 4+ 97 TELRTYTI 38 577-591 d 12 5 4+ LNRKAID 101 CRILGPDC 39601-615 b 80 41 4+ CIEPHDW 105 QIIHDFID 40 625-639 b 28 23 4+ NPLPNQD110 GIGITGII 41 655-669 b 27 19 8+ IAIIALL “Epitope-containing peptideswere identified by IFNγ ELISPOT (≥10 SFC/10⁶ splenocytes AND ≥80%response rate) and then confirmed by FACS (≥3-5 × 10⁴ CD3+ cells wereacquired). Responses for each were further characterized by FACS(expression of CD4 and/or CD8 by CD3+/CD44+/IFNγ+ cells). PredictedCD8+ epitopes arc underlined (best consensus % rank by IEDB) andpreviously-described epitopes are referenced. Immunodominant epitopesare displayed (*).

TABLE 3 Plasmid Vaccine pEBOZ GP sequence ZEBOV CON SEQ FACS Peptide IDELISPOT T cell Number Sequence NO: Position H-2 AVE ±SEM restr. 6FSIPLGVI 42 31-45 d 78 31 8+ HNSTLQV 15 RWGFRSGV 43 85-99 b 44 12 4+PPKVVNY 19 YNLEIKKP 44 109-123 b 29 12 4+ DGSECLP 24 HKVSGTGPC 45139-153 d 484 85 8+ AGDFAF 27 GAFFLYDR 46 157-171 d 72 18 8+ LASTVIY 30TFAEGVVA 47 175-189 d 581 85 4+ FLILPQA 32 PQAKKDFFS 48 187-201 b 18 64+ SHPLRE 33 FFSSHPLR 49 193-207 b 21 8 4+ EPVNATE 40 EVDNLTYV 50235-249 d 32 17 4+ QLESRFT 41 YVQLESRF 51 241-255 d 97 23 4+ TPQFLLQ 48TTIGEWAF 52 283-297 d 219 70 4+ WETKKNL 49 AFWETKKNL 53 289-303 d 32 154+ TRKIRS 50 KNLTRKIR 54 295-309 d 105 37 4+ SEELSFT 60 SQGREAAV 55355-369 b 16 7 4+ SHLTTLA 65 DNSTHNTP 56 385-399 d 29 18 4+ VYKLDIS 66TPVYKLDI 57 391-405 d 371 118 4+ SEATQVE 71 PPATTAAGP 58 421-435 b 21 84+ PKAENT 84 TRREAIVN 59 499-513 b 12 5 8+ AQPKCNP 89 LAWIPYFG 60529-543 b 93 8 8+/ PAAEGIY 4+ 97 TELRTFSI 61 577-591 b/d 14/ 4/ 8+LNRKAID 82 42 101 CHILGPDC 62 601-615 b 96 62 4+ CIEPHDW“Epitope-containing peptides were identified by IFNγ ELISPOT (≥10SFC/106 splenocytes AND ≥80% response rate) and then confirmed by FACS(≥3-5 × 104 CD3+ cells were acquired). Responses for each were furthercharacterized by FACS (expression of CD4 and/or CD8 byCD3+/CD44+/IFNγ+ cells). Predicted CD8+ epitopes arc underlined (bestconsensus % rank by IEDB) and previously-described epitopes arereferenced. Immunodominant epitopes are displayed (*).

TABLE 4 Plasmid Vaccine pMARV GP sequence MARV ANGBest con % rank (IEDB) SEQ CD8+ (≤0.5) CD4+ (<25) Peptide IDPreviously defined (80% Blast; Allele Number Sequence NO: D^(b) K^(b)D^(d) K^(d) L^(d) I-A^(b) I-A^(d) I-E^(d) 3 IQGVKTLP 7 12.1 ILEIASN 5ASNIQPQN 8 0.4 VDSVCSG 12 SKRWAFRA 9 0.8 GVPPKNV 27 GKVFTEGN 10 12.9IAAMIVN 28 GNIAAMIV 11 0.2 3.9 H-2^(d) NKTVHKM class I GNIAAMIV 12 0.23.9 NKTVHKM 29 IVNKTVHK 13 17.2 MIFSRQG 30 HKMIFSRQ 14 GQGYRHM 31RQGQGYRH 15 0.1 23.9 MNLTSTN 32 RHMNLTST 16 NKYWTSS 65 LPTENPTT 17 24.0AKSTNST 71 PNSTAQHL 18 7.5 VYFRRKR 72 HLVYFRRK 19 0.3 8.3 RNILWRE 89GLSWIPFF 20 7.0 GPGIEGL 92 GLIKNQNN 21 LVCRLRR 93 NNLVCRLR 22 13.3RLANQTA 97 TTEERTFS 23 0.1 0.4 LINRHAI 99 HAIDFLLA 24 21.8 RWGGTCK 101TCKVLGPD 25 0.4 CCIGIED “Epitope-containing peptides were identified byIFNγ ELISPOT (≥10 SFC/10⁶ splenocytes AND ≥80% response rate) and thenconfirmed by FACS (≥3-5 × 10⁴ CD3+ cells were acquired). Responses foreach were further characterized by FACS (expression of CD4 and/or CD8 byCD3+/CD44+/IFNγ+ cells). Predicted CD8+ epitopes are underlined (bestconsensus % rank by IEDB) and previously-described epitopes arereferenced. Immunodominant epitopes are displayed (*).

TABLE 5 Plasmid Vaccine pEBOS GP sequence SUDV CONBest con % rank (IEDB) SEQ CD8+ (≤0.5) CD4+ (<25) Peptide IDPreviously defined (80% Blast; Allele Number Sequence NO: D^(b) K^(b)D^(d) K^(d) L^(d) I-A^(b) I-A^(d) I-E^(d) 4 FFVWVIILFQKAFSM 26 0.4 15RWGFRSGVPPKVVSY 27 1.2 19 YNLEIKKPDGSECLP 28 24 HKAQGTGPCPGDYAF 29 0.327 GAFFLYDRLASTVIY 30 0.3 21.1 23.4 H-2^(b) class I 30 NFAEGVIAFLILAKP31 0.1 36 SYYATSYLEYEIENF 32 0.4 0.3 0.1 41 FVLLDRPHTPQFLFQ 33 0.1 78NITTAVKTVLPQEST 34 7.2 82 TGILGSLGLRKRSRR 35 17.2 83 LGLRKRSRRQVNTRA 3689 IAWIPYFGPGAEGIY 37 0.1 3.0 97 TELRTYTILNRKAID 38 0.1 18.5 21.2 101CRILGPDCCIEPHDW 39 105 QIIHDFIDNPLPNQD 40 0.3 110 GIGITGIIIAIIALL 41“Epitope-containing peptides were identified by IFNγ ELISPOT (≥10SFC/10⁶ splenocytes AND ≥80% response rate) and then confirmed by FACS(≥3-5 × 10⁴ CD3+ cells were acquired). Responses for each were furthercharacterized by FACS (expression of CD4 and/or CD8 byCD3+/CD44+/IFNγ+ cells). Predicted CD8+ epitopes are underlined (bestconsensus % rank by IEDB) and previously-described epitopes arereferenced. Immunodominant epitopes are displayed (*).

TABLE 6 Plasmid Vaccine pEBOZ GP sequence ZEBOV CONBest con % rank (IEDB) SEQ CD8+ (≤0.5) CD4+ (<25) Peptide IDPreviously defined (80% Blast; Allele Number Sequence NO: D^(b) K^(b)D^(d) K^(d) L^(d) I-A^(b) I-A^(d) I-E^(d) 6 FSIPLGVIHNSTLQV 42 0.2H-2^(d) class I 15 RWGFRSGVPPKVVNY 43 1.2 H-2^(d) class I 19YNLEIKKPDGSECLP 44 24 HKVSGTGPCAGDFAF 45 0.1 14.9 27 GAFFLYDRLASTVIY 460.3 21.1 23.4 30 TFAEGVVAFLILPQA 47 0.2 21.6 32 PQAKKDFFSSHPLRE 48 0.10.4 16.4 33 FFSSHPLREPVNATE 49 14.7 40 EVDNLTYVQLESRFT 50 0.4 19.6H-2^(b) class I 41 YVQLESRFTPQFLLQ 51 H-2^(k) class I 48 TTIGEWAFWETKKNL52 12.9 49 AFWETKKNLTRKIRS 53 22.9 50 KNLTRKIRSEELSFT 54 22.7 60SQGREAAVSHLTTLA 55 0.3 23.1 3.9 65 DNSTHNTPVYKLDIS 56 66 TPVYKLDISEATQVE57 22.6 5.5 71 PPATTAAGPPKAENT 58 2.1 84 TRREAIYNAQPKCNP 59 0.3 14.6 7.989 LAWIPYFGPAAEGIY 60 0.1 0.8 97 TELRTFSILNRKAID 61 0.1 22.2 101CHILGPDCCIEPHDW 62 “Epitope-containing peptides were identified byIFNγ ELISPOT (≥10 SFC/10⁶ splenocytes AND ≥80% response rate) and thenconfirmed by FACS (≥3-5 × 10⁴ CD3+ cells were acquired). Responses foreach were further characterized by FACS (expression of CD4 and/or CD8 byCD3+/CD44+/IFNγ+ cells). Predicted CD8+ epitopes are underlined (bestconsensus % rank by IEDB) and previously-described epitopes arereferenced. Immunodominant epitopes are displayed (*).

1.-28. (canceled)
 29. An isolated nucleic acid molecule comprising anucleic acid encoding a consensus Zaire ebolavirus envelope glycoproteinimmunogen (ZEBOVCON2), wherein the ZEBOVCON2 comprises an amino acidsequence as set forth in SEQ ID NO:68, or a fragment of an amino acidsequence comprising at least 600 consecutive amino acid residues of SEQID NO:68.
 30. The isolated nucleic acid molecule of claim 29, whereinthe fragment of an amino acid sequence that is at least 95% homologousto SEQ ID NO:68 comprises at least 600 amino acids, at least 630 aminoacids, or at least 660 amino acids.
 31. The isolated nucleic acidmolecule of claim 29, ZEBOVCON2 is linked to an IgE signal peptide. 32.The isolated nucleic acid molecule of claim 29, wherein the nucleic acidencoding ZEBOVCON2 comprises a nucleic acid sequence at least 95%homologous SEQ ID NO:70, or a fragment thereof.
 33. The isolated nucleicacid molecule of claim 29, wherein the nucleic acid molecule is aplasmid.
 34. The isolated nucleic acid molecule of claim 29, formulatedfor delivery to an individual using electroporation.
 35. A compositioncomprising a nucleic acid molecule of claim
 29. 36. The composition ofclaim 35, wherein the fragment of an amino acid sequence that is atleast 95% homologous to SEQ ID NO:68 comprises at least 600 amino acids,at least 630 amino acids, or at least 660 amino acids.
 37. Thecomposition of claim 35, wherein the ZEBOVCON2 is linked to an IgEsignal peptide.
 38. The composition of claim 35, wherein the nucleicacid encoding ZEBOVCON2 comprises a nucleic acid sequence at least 95%homologous SEQ ID NO:70, or a fragment thereof.
 39. The composition ofclaim 35, wherein the nucleic acid molecule is a plasmid.
 40. Thecomposition of claim 35, formulated for delivery to an individual usingelectroporation.
 41. The composition of claim 35 further comprisingnucleic acid sequences that encode one or more proteins selected fromthe group consisting of: IL-12, IL-15 and IL-28.
 42. A method ofinducing an immune response against a Filovirus comprising administeringthe nucleic acid molecule of claim 29 to an individual in an amounteffective to induce an immune response in said individual.
 43. Themethod of claim 42, wherein the method induces an immune responseagainst an Ebolavirus.
 44. A method of inducing an immune responseagainst a Filovirus comprising administering the composition of claim 35to an individual in an amount effective to induce an immune response insaid individual.
 45. The method of claim 44, wherein the method inducesan immune response against an Ebolavirus.
 46. A method of treating anindividual who has been diagnosed with Ebolavirus comprisingadministering a therapeutically effective amount of the nucleic acidmolecule of claim 29 to an individual.
 47. A method of treating anindividual who has been diagnosed with Ebolavirus, the method comprisingadministering a therapeutically effective amount of the composition ofclaim 35 to an individual.